Cationic oil-in-water emulsions

ABSTRACT

This invention generally relates to cationic oil-in-water emulsions that contain high concentrations of cationic lipids and have a defined oil:lipid ratio. The cationic lipid can interact with the negatively charged molecule thereby anchoring the molecule to the emulsion particles. The cationic emulsions described herein are useful for delivering negatively charged molecules, such as nucleic acid molecules to cells, and for formulating nucleic acid-based vaccines.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/130,886, filed Apr. 14, 2014, which is the U.S. National Stage ofInternational Application No. PCT/US2012/045845, filed Jul. 6, 2012 andpublished in English, which claims the benefit of U.S. ProvisionalApplication No. 61/505,109, filed Jul. 6, 2011, U.S. ProvisionalApplication No. 61/545,936, filed Oct. 11, 2011, and U.S. ProvisionalApplication No. 61/585,641, filed Jan. 11, 2012; the entire contents ofeach of the foregoing patent applications is incorporated herein byreference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 5, 2012, isnamed PAT54691.txt and is 424,203 bytes in size.

BACKGROUND OF THE INVENTION

Nucleic acid therapeutics have promise for treating diseases rangingfrom inherited disorders to acquired conditions such as cancer,infectious disorders (AIDS), heart disease, arthritis, andneurodegenerative disorders (e.g., Parkinson's and Alzheimer's). Notonly can functional genes be delivered to repair a genetic deficiency orinduce expression of exogenous gene products, but nucleic acid can alsobe delivered to inhibit endogenous gene expression to provide atherapeutic effect. Inhibition of gene expression can be mediated by,e.g., antisense oligonucleotides, double-stranded RNAs (e.g., siRNAs,miRNAs), or ribozymes.

A key step for such therapy is to deliver nucleic acid molecules intocells in vivo. However, in vivo delivery of nucleic acid molecules, inparticular RNA molecules, faces a number of technical hurdles. First,due to cellular and serum nucleases, the half life of RNA injected invivo is only about 70 seconds (see, e.g., Kurreck, Eur. J. Bioch.270:1628-44 (2003)). Efforts have been made to increase stability ofinjected RNA by the use of chemical modifications; however, there areseveral instances where chemical alterations led to increased cytotoxiceffects or loss of or decreased function. In one specific example, cellswere intolerant to doses of an RNAi duplex in which every secondphosphate was replaced by phosphorothioate (Harborth, et al, AntisenseNucleic Acid Drug Rev. 13(2): 83-105 (2003)). As such, there is a needto develop delivery systems that can deliver sufficient amounts ofnucleic acid molecules (in particular RNA molecules) in vivo to elicit atherapeutic response, but that are not toxic to the host.

Nucleic acid based vaccines are an attractive approach to vaccination.For example, intramuscular (IM) immunization of plasmid DNA encoding forantigen can induce cellular and humoral immune responses and protectagainst challenge. DNA vaccines offer certain advantages overtraditional vaccines using protein antigens, or attenuated pathogens.For example, as compared to protein vaccines, DNA vaccines can be moreeffective in producing a properly folded antigen in its nativeconformation, and in generating a cellular immune response. DNA vaccinesalso do not have some of the safety problems associated with killed orattenuated pathogens. For example, a killed viral preparation maycontain residual live viruses, and an attenuated virus may mutate andrevert to a pathogenic phenotype.

One limitation of nucleic acid based vaccines is that large doses ofnucleic acid are generally required to obtain potent immune responses innon-human primates and humans. Therefore, delivery systems and adjuvantsare required to enhance the potency of nucleic acid based vaccines.Various methods have been developed for introducing nucleic acidmolecules into cells, such as calcium phosphate transfection, polyprenetransfection, protoplast fusion, electroporation, microinjection andlipofection.

Cationic lipids have been formulated as liposomes to deliver genes intocells. In addition, cationic lipid emulsions have been developed todeliver DNA molecules into cells. See, e.g., Kim, et al., InternationalJournal of Pharmaceutics, 295, 35-45 (2005).

Ott et al. (Journal of Controlled Release, volume 79, pages 1-5, 2002)describes an approach involving a cationic sub-micron emulsion as adelivery system/adjuvant for DNA. The sub-micron emulsion approach isbased on MF59, a potent squalene in water adjuvant that is a componentof commercially approved product Fluad®.1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) was used to facilitateintracellular delivery of plasmid DNA.

Yi et al. (Pharmaceutical Research, 17, 314-320 (2000)) disclosescationic oil-in-water emulsions that used soybean oil and DOTAP as thecationic lipid. Cholesterol, DOPE, and polymeric lipids were alsoincluded in some of the emulsions. The emulsions were shown to enhancethe efficiency of in vitro transfection of DNA in the presence of up to90% serum. The average size of the emulsion particles ranged from 181 nmto 344 nm, and the particle size increased after the emulsions werediluted in PBS buffer.

Kim et al. (Pharmaceutical Research, vol. 18, pages 54-60, 2001) andChung et al. (Journal of Controlled Release, volume 71, pages 339-350,2001) disclose various oil-in-water emulsions that were used to enhancein vitro and in vivo transfection efficiency of DNA molecules. Among thecationic lipids tested, DOTAP formed the most stable and efficientemulsion for DNA delivery. Among the oils tested, squalene, lightmineral oil, and jojoba bean oil formed stable emulsions with smallparticles. The efficiencies of in vitro transfection were shown tocorrelate to the stability of the emulsions (e.g., the emulsionformulated by squalene at 100 mg/mL and DOTAP at 24 mg/mL showed high invitro transfection efficiency). The emulsions were prepared by firstmixing the cationic lipid with water to form a liposome suspension (bysonication). Liposomes were then added to the oil (such as squalene) andthe mixture was sonicated to form an oil-in-water emulsion.

RNA molecules encoding an antigen or a derivative thereof may also beused as vaccines. RNA vaccines offer certain advantages as compared toDNA vaccines. However, compared with DNA-based vaccines, relativelyminor attention has been given to RNA-based vaccines. RNAs are highlysusceptible to degradation by nucleases when administered as atherapeutic or vaccine. Additionally, RNAs are not actively transportedinto cells. See, e.g., Vajdy, M., et al., Mucosal adjuvants and deliverysystemsfor protein-, DNA-and RNA-based vaccines, Immunol Cell Biol,2004. 82(6): p. 617-27.

Therefore, there is a need to provide delivery systems for nucleic acidmolecules or other negatively charged molecules. The delivery systemsare useful for nucleic acid-based vaccines, in particular RNA-basedvaccines.

SUMMARY OF THE INVENTION

The invention relates to cationic oil-in-water emulsions that containhigh concentrations of cationic lipids and have a defined oil:lipidratio. The oil and cationic lipid are separate components of theemulsions, and preferably the oil is not ionic. The cationic lipid caninteract with the negatively charged molecule thereby anchoring themolecule to the emulsion particles. The cationic emulsions describedherein are useful for delivering negatively charged molecules, such asnucleic acid molecules (e.g., an RNA molecule encoding an antigen), tocells, and for formulating nucleic acid-based vaccines.

In one aspect, the invention provides an oil-in-water emulsioncomprising particles that are dispersed in an aqueous continuous phase,wherein the emulsion is characterized by: (a) the average diameter ofsaid particles is from about 80 nm to 180 nm in diameter; (b) theemulsion comprises an oil and a cationic lipid, wherein (i) the ratio ofoil:cationic lipid (mole:mole) is at least about 8:1 (mole:mole), (ii)the concentration of cationic lipid in said emulsion is at least about2.5 mM, and (iii) with the proviso that the cationic lipid is notDC-Cholesterol. Preferably, the oil-in-water emulsion is stable. In someembodiments, the ratio of oil:lipid (mole:mole) is from about 10:1(mole:mole) to about 43:1 (mole:mole). The oil in water emulsion cancontain from about 0.2% to about 8% (w/v) oil. In some embodiments, theoil is squalene or squalane.

In another aspect, the invention provides an oil-in-water emulsioncomprising particles that are dispersed in an aqueous continuous phase,wherein the emulsion is characterized by: (a) the average diameter ofsaid particles is from about 80 nm to 180 nm in diameter; (b) theemulsion comprises an oil and a cationic lipid, wherein (i) the ratio ofoil:cationic lipid (mole:mole) is at least about 4:1 (mole:mole), (ii)the concentration of cationic lipid in said emulsion is at least about2.5 mM, (iii) the oil is present from about 0.2% to about 8% (w/v); and(iv) with the proviso that the cationic lipid is not DC-Cholesterol.Preferably, the oil-in-water emulsion is stable. In some embodiments,the ratio of oil:lipid (mole:mole) is from about 4:1 (mole:mole) toabout 43:1 (mole:mole). In some embodiments, the oil is squalene orsqualane. In some embodiments, the oil is present from 0.6% to 4% (w/v).In some embodiments, the oil is present from about 1% to about 3.2%(w/v).

The oil-in-water emulsion of this aspect can further comprise asurfactant, such as a nonionic surfactant. Preferably, the surfactant isnot a Polyethylene Glycol (PEG)-lipid. The surfactant can be present inan amount from about 0.01% to about 2.5% (w/v). In some embodiments, thesurfactant is SPAN85 (Sorbtian Trioleate), Tween 80 (polysorbate 80), ora combination thereof. In some embodiments, the oil-in-water emulsioncontains equal amounts of SPAN85 (Sorbtian Trioleate) and Tween 80(polysorbate 80), for example 0.5% (w/v) of each.

Preferably the head group of the cationic lipid comprises a quaternaryamine. For example, in some embodiments the cationic lipid is selectedfrom the group consisting of:1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP),1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC),N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), and N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).

In some embodiments, the emulsion is characterized by: (a) the averagediameter of the emulsion particles is from about 80 nm to 180 nm indiameter; (b) the emulsion comprises an oil and DOTAP, wherein (i) theratio of oil:DOTAP (mole:mole) is at least about 8:1 (mole:mole), and(ii) the concentration of DOTAP in said emulsion is at least about 2.58mM (1.8 mg/mL), or from about 2.58 mM (1.8 mg/mL) to about 7.16 mM (5mg/mL). The oil can be squalene or squalane.

In some embodiments, the emulsion is characterized by: (a) the averagediameter of the emulsion particles is from about 80 nm to 180 nm indiameter; (b) the emulsion comprises an oil and DOTAP, wherein (i) theratio of oil:DOTAP (mole:mole) is at least about 4:1 (mole:mole), (ii)the concentration of DOTAP in said emulsion is at least about 2.58 mM(1.8 mg/mL), and (iii) the oil is present from about 0.2% to about 8%(w/v). In some embodiments, the oil is squalene or squalane. In someembodiments, the concentration of DOTAP from about 2.58 mM (1.8 mg/mL)to about 7.16 mM (5 mg/mL). In some embodiments, the oil is present from0.6% to 4% (w/v). In some embodiments, the oil is present from about 1%to about 3.2% (w/v).

The invention also provides a method for preparing an oil-in-wateremulsion comprising particles that are dispersed in an aqueouscontinuous phase, wherein the emulsion is characterized by: (a) theaverage diameter of said particles is from about 80 nm to 180 nm indiameter; (b) the emulsion comprises an oil and a cationic lipid,wherein (i) the ratio of oil:cationic lipid (mole:mole) is at leastabout 8:1 (mole:mole), (ii) the concentration of cationic lipid in saidemulsion is at least about 2.5 mM, and (iii) with the proviso that thecationic lipid is not DC-Cholesterol, the method comprises (a) directlydissolving the cationic lipid in the oil to form an oil phase; (b)providing an aqueous phase of the emulsion; and (c) dispersing the oilphase in the aqueous phase by homogenization. The oil can be heated to atemperature between about 30° C. to about 65° C. to facilitatedissolution of the cationic lipid in the oil. Higher temperatures mayalso be used, as long as there is no significant degradation of oil orthe cationic lipid.

The invention also provides a method for preparing an oil-in-wateremulsion comprising particles that are dispersed in an aqueouscontinuous phase, wherein the emulsion is characterized by: (a) theaverage diameter of said particles is from about 80 nm to 180 nm indiameter; (b) the emulsion comprises an oil and a cationic lipid,wherein (i) the ratio of oil:cationic lipid (mole:mole) is at leastabout 4:1 (mole:mole), (ii) the concentration of cationic lipid in saidemulsion is at least about 2.5 mM, (iii) the oil is present from about0.2% to about 8% (w/v); and (iv) with the proviso that the cationiclipid is not DC-Cholesterol, the method comprises (a) directlydissolving the cationic lipid in the oil to form an oil phase; (b)providing an aqueous phase of the emulsion; and (c) dispersing the oilphase in the aqueous phase by homogenization. The oil can be heated to atemperature between about 30° C. to about 65° C. to facilitatedissolution of the cationic lipid in the oil. Higher temperatures mayalso be used, as long as there is no significant degradation of oil orthe cationic lipid.

In another aspect, the invention provides a composition comprising anucleic acid molecule (preferably an RNA molecule) complexed with aparticle of a cationic oil-in-water emulsion, wherein the particlecomprises an oil that is in liquid phase at 25° C., and a cationiclipid; and (i) the ratio of oil:lipid (mole:mole) is at least about 8:1(mole:mole); (ii) the concentration of cationic lipid in saidcomposition is at least about 1.25 mM; and (iii) with the proviso thatthe cationic lipid is not DC-Cholesterol. Preferably, the averagediameter of the emulsion particles is from about 80 nm to 180 nm, orabout 80 nm to 150 nm, or about 80 nm to about 130 nm, and the NIP ratioof the composition is at least about 4:1, or from about 4:1 to about20:1, or from about 4:1 to about 15:1. In certain embodiments, the ratioof oil:lipid (mole:mole) is from about 10:1 (mole:mole) to about 43:1(mole:mole). The oil in water emulsion can contain from about 0.1% toabout 5% (w/v) oil. In some embodiments, the oil is squalene orsqualane.

In another aspect, the invention provides a composition comprising anucleic acid molecule (preferably an RNA molecule) complexed with aparticle of a cationic oil-in-water emulsion, wherein the particlecomprises an oil that is in liquid phase at 25° C., and a cationiclipid; and (i) the ratio of oil:lipid (mole:mole) is at least about 4:1(mole:mole); (ii) the concentration of cationic lipid in saidcomposition is at least about 1.25 mM; (iii) the oil is present fromabout 0.1% to about 4% (w/v); and (iv) with the proviso that thecationic lipid is not DC-Cholesterol. Preferably, the average diameterof the emulsion particles is from about 80 nm to 180 nm, or about 80 nmto 150 nm, or about 80 nm to about 130 nm, and the NIP ratio of thecomposition is at least about 4:1, or from about 4:1 to about 20:1, orfrom about 4:1 to about 15:1. In certain embodiments, the ratio ofoil:lipid (mole:mole) is from about 4:1 (mole:mole) to about 43:1(mole:mole). In some embodiments, the oil is squalene or squalane. Insome embodiments, the oil is present from 0.6% to 4% (w/v). In someembodiments, the oil is present from about 1% to about 3.2% (w/v).

The oil-in-water emulsion of this aspect can further comprise asurfactant, such as a nonionic surfactant. Preferably, surfactant is nota Polyethylene Glycol (PEG)-lipid. The surfactant can be present in anamount from about 0.005% to about 1.25% (w/v). In some embodiments, thesurfactant is SPAN85 (Sorbtian Trioleate), Tween 80 (polysorbate 80), ora combination thereof. In some embodiments, the oil-in-water emulsioncontains equal amounts of SPAN85 (Sorbtian Trioleate) and Tween 80(polysorbate 80), for example 0.25% or 0.5% (w/v) of each.

Preferably the head group of the cationic lipid comprises a quaternaryamine. For example, in some embodiments the cationic lipid is selectedfrom the group consisting of:1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP),1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC),N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), and N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).

In some embodiments, the invention provides a composition comprising anucleic acid molecule (preferably an RNA molecule) complexed with aparticle of a cationic oil-in-water emulsion, wherein the particlecomprises an oil that is in liquid phase at 25° C. and DOTAP; and (i)the ratio of oil:DOTAP (mole:mole) is at least about 8:1 (mole:mole);(ii) the concentration of DOTAP in said composition is at least about1.29 mM, or from about 1.29 mM (0.9 mg/mL) to about 3.58 mM (2.5 mg/mL).The oil can be squalene or squalane. Optionally, the N/P ratio is atleast 4:1.

In preferred embodiments, the composition is buffered (e.g., with acitrate buffer, succinate buffer, acetate buffer) and has a pH of about6.0 to about 8.0, preferably about 6.2 to about 6.8. The composition canfurther comprise an inorganic salt, and the concentration of inorganicsalt is preferably no greater than 30 mM. Optionally, the compositioncan further comprise a nonionic tonicifying agent, and preferably isisotonic.

The invention also provides a method for preparing a compositioncomprising a nucleic acid molecule (preferably an RNA molecule)complexed with a particle of a cationic oil-in-water emulsion,comprising: (i) providing an oil-in-water emulsion as described herein;(ii) providing an aqueous solution comprising the RNA molecule; and(iii) combining the oil-in-water emulsion of (i) and the aqueoussolution of (ii), thereby preparing the composition. In someembodiments, the cationic oil-in-water emulsion and RNA solution arecombined at about 1:1 (v/v) ratio. The aqueous solution comprising theRNA molecule is preferably buffered (e.g., with a citrate buffer,succinate buffer, acetate buffer), can contain a inorganic salt (e.g.NaCl), which is preferably present at about 20 mM or less. In oneembodiment, the aqueous solution comprising the RNA molecule contains 2mM citrate buffer and 20 mM NaCl. Optionally, the aqueous solutioncomprising the RNA molecule further comprises an nonionic tonicifyingagent, and is isotonic. In one embodiment, the aqueous solution furthercomprises about 560 mM sucrose. Optionally, the aqueous solutioncomprising the RNA molecule further comprises a polymer or nonionicsurfactant, such as Pluronic® F127, at from about 0.05% to about 20%(w/v).

In another aspect, the invention provides an oil-in-water emulsioncomprising particles that are dispersed in an aqueous continuous phase,wherein the emulsion comprises an oil and a cationic lipid, the averagediameter of said particles is from about 80 nm to 180 nm, the oil ispresent from 0.6% to 4% (w/v); and the concentration of cationic lipidin said emulsion is at least about 1.25 mM. Preferably, the oil-in-wateremulsion is stable. In some embodiments, the concentration of cationiclipid in said emulsion is at least about 2.5 mM. In some embodiments,the oil is squalene or squalane.

The oil-in-water emulsion of this aspect can further comprise asurfactant, such as a nonionic surfactant. Preferably, surfactant is nota Polyethylene Glycol (PEG)-lipid. The surfactant can be present in anamount from about 0.01% to about 2.5% (w/v). In some embodiments, thesurfactant is SPAN85 (Sorbtian Trioleate), Tween 80 (polysorbate 80), ora combination thereof. In some embodiments, the oil-in-water emulsioncontains equal amounts of SPAN85 (Sorbtian Trioleate) and Tween 80(polysorbate 80), for example 0.25% or 0.5% (w/v) of each.

Preferably the head group of the cationic lipid comprises a quaternaryamine. For example, in some embodiments the cationic lipid is selectedfrom the group consisting of:1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP),1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC),N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), and N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).

The invention provides a composition comprising a nucleic acid molecule(preferably an RNA molecule) complexed with a particle of anoil-in-water emulsion that contains particles that are dispersed in anaqueous continuous phase, wherein the emulsion comprises an oil and acationic lipid, the average diameter of said particles is from about 80nm to 180 nm, the oil is present from 0.6% to 4% (w/v); and theconcentration of cationic lipid in said emulsion is at least about 1.25mM. Preferably, the oil-in-water emulsion is stable. In someembodiments, the concentration of cationic lipid in said emulsion is atleast about 2.5 mM. In some embodiments, the oil s squalene or squalane.Preferably, the N/P ratio of the composition is at least about 4:1.

In preferred embodiments, the composition is buffered (e.g., with acitrate buffer, succinate buffer, acetate buffer) and has a pH of about6.0 to about 8.0, preferably about 6.2 to about 6.8. The composition canfurther comprise an inorganic salt, and the concentration of inorganicsalt is preferably no greater than 30 mM. Optionally, the compositioncan further comprise a nonionic tonicifying agent, and preferably isisotonic.

The invention also relates to a method of generating an immune responsein a subject, comprising administering to a subject in need thereof thecomposition as described herein. Preferably the amount of the cationiclipid administered to the subject (as a component of the composition) ina single administration is no more than about 30 mg. In particularembodiments, the cationic lipid is DOTAP and the total amount of DOTAPadministered to the subject in a single administration is no more thanabout 24 mg, or no more than about 4 mg.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of pentacistronic RNA replicons, A526, A527, A554,A555 and A556, that encode five CMV proteins. Subgenomic promoters areshown by arrows, other control elements are labeled.

FIG. 2 is a fluorescence histogram showing that BHKV cells transfectedwith the A527 RNA replicon express the gH/gL/UL128/UL130/UL131pentameric complex. Cell stain was performed using an antibody thatbinds a conformational epitope present on the pentameric complex.

FIG. 3 is a schematic and graph. The schematic shows bicistronic RNAreplicons, A160 and A531-A537, that encode CMV gH and gL. The graphshows neutralizing activity of immune sera from mice immunized with VRPsthat contained the replicons.

FIG. 4 is a graph showing anti-VZV protein antibody response in immunesera from mice immunized with monocistronic RNA replicons that encodedVZV proteins or bicistronic RNA replicons that encoded VZV gE and gI, orgH and gL. The mice were immunized with 7 μg RNA formulated with CMF32.

DETAILED DESCRIPTION OF THE INVENTION 1. Overview

This invention generally relates to cationic oil-in-water emulsions thatcontain high concentrations of cationic lipids and have a definedoil:cationic lipid ratio. The oil and cationic lipid are separatecomponents of the emulsions, and preferably the oil is not ionic. Thecationic lipid can interact with a negatively charged molecule, such asa nucleic acid, thereby anchoring the negatively charged molecule to theemulsion particles. The cationic emulsions described herein are usefulfor delivering negatively charged molecules, such as nucleic acidmolecules (e.g., an RNA molecule encoding a protein or peptide, smallinterfering RNA, self-replicating RNA, and the like), to cells in vivo,and for formulating nucleic acid-based vaccines.

In particular, the present invention is based on the discovery thatstable cationic oil-in-water emulsions that contain high concentrationsof cationic lipids and have a defined oil:cationic lipid ratio can besuccessfully made. Emulsions that contain high concentrations ofcationic lipids allow more negatively charged molecules (such as RNAmolecules) to be formulated with emulsion particles, thereby increasingthe efficiency of delivery. In particular, for many therapeutics such asvaccines small volumes (e.g., 0.5 mL per dose) are preferred foradministration. Emulsions that contain high concentrations of cationiclipids and have a defined oil:cationic lipid ratio, as described herein,will allow for the delivery of a higher dose of RNA within a specifiedvolume.

In preferred embodiments, an RNA molecule is complexed with a particleof the oil-in-water emulsion. The complexed RNA molecule is stabilizedand protected from RNase-mediated degradation, and is more efficientlytaken up by cells relative to free (“naked”) RNA.

In addition, when the RNA is delivered to induce expression of anencoded protein, such as in the context of an RNA vaccine, emulsionsthat contain high concentrations of cationic lipids can increase theamount of RNA molecules that are complexed with emulsion particles. Asmore RNA molecules are delivered to host cells, higher amount of theencoded protein antigen is produced, which in turn enhances the potencyand immunogenicity of the RNA vaccine. Finally, the immunogenicity ofthe encoded protein can be enhanced due to adjuvant effects of theemulsion. Therefore, in addition to more efficient delivery of anegatively charged molecule (e.g., an RNA molecule that encodes anantigen), the cationic emulsions can also enhance the immune responsethrough adjuvant activity. For example, as described and exemplifiedherein, formulations in which RNA molecules (encoding respiratorysyncytial virus (RSV) F protein) were complexed with high-DOTAPemulsions generated higher immune responses in a mouse model and acotton rat model of RSV, as compared to RNA molecules complexed withlow-DOTAP emulsions.

Accordingly, in one aspect, the invention provides an oil-in-wateremulsion comprising particles that are dispersed in an aqueouscontinuous phase, wherein the emulsion is characterized by: (a) theaverage diameter of said particles is from about 80 nm to 180 nm; (b)the emulsion comprises an oil and a cationic lipid, wherein (i) theratio of oil:cationic lipid (mole:mole) is at least about 8:1(mole:mole), (ii) the concentration of cationic lipid in said emulsionis at least about 2.5 mM, and (iii) the cationic lipid is notDC-Cholesterol.

In another aspect, the invention provides an oil-in-water emulsioncomprising particles that are dispersed in an aqueous continuous phase,wherein the emulsion is characterized by: (a) the average diameter ofsaid particles is from about 80 nm to 180 nm; (b) the emulsion comprisesan oil and a cationic lipid, wherein (i) the ratio of oil:cationic lipid(mole:mole) is at least about 4:1 (mole:mole), (ii) the concentration ofcationic lipid in said emulsion is at least about 2.5 mM, (iii) the oilis present from about 0.2% to about 8% (w/v); and (iv) with the provisothat the cationic lipid is not DC-Cholesterol.

The cationic emulsion may further comprise a surfactant (e.g., Tween 80,SPAN85, or a combination thereof).

In another aspect, the invention also provides several specificformulations of cationic oil-in-water emulsions that contain highconcentrations of cationic lipids and can be used to deliver negativelycharged molecules.

In another aspect, the invention provides a method of preparing anoil-in-water emulsion, comprising: (1) directly dissolving a cationiclipid in an oil to form an oil phase; (2) providing an aqueous phase ofthe emulsion; and (3) dispersing the oil phase in the aqueous phase(e.g., by homogenization). If desired, the oil may be heated to atemperature between about 30° C. to about 65° C. to facilitate thedissolving of the lipid in the oil. Preferably, the ratio ofoil:cationic lipid (mole:mole) in the oil phase is at least about 8:1(mole:mole), and alternatively or in addition, the average diameter ofsaid particles is from about 80 nm to 180 nm, and/or the concentrationof cationic lipid in the oil phase is at least about 5 mM.

In another aspect, the invention provides a method of preparing acomposition that comprises a negatively charged molecule (such as an RNAmolecule) complexed with a particle of a cationic oil-in-water emulsion,comprising: (i) providing an oil-in-water emulsion as described herein;(ii) providing an aqueous solution comprising the RNA molecule; and(iii) combining the aqueous solution of (ii) and the oil-in-wateremulsion of (i), thereby preparing the composition. If desired, theaqueous solution comprising the RNA molecule may comprise a salt (e.g.,NaCl), a buffer (e.g., a citrate buffer), a nonionic tonicifying agent(e.g., sucrose, trehalose, sorbitol, or dextrose), a polymer (e.g.,Pluronic® F127), or any combination thereof.

The cationic emulsions of the invention can be used to deliver anegatively charge molecule, such as a nucleic acid (e.g., RNA). Thecompositions may be administered to a subject in need thereof togenerate or potentiate an immune response. The compositions can also beco-delivered with another immunogenic molecule, immunogenic compositionor vaccine to enhance the effectiveness of the induced immune response.

2. Definitions

The term “about”, as used here, refers to +/−5% of a value.

An “antigen” refers to a molecule containing one or more epitopes(either linear, conformational or both).

A “buffer” refers to an aqueous solution that resists changes in the pHof the solution.

As used herein, “nucleotide analog” or “modified nucleotide” refers to anucleotide that contains one or more chemical modifications (e.g.,substitutions) in or on the nitrogenous base of the nucleoside (e.g.,cytosine (C), thymine (T) or uracil (U)), adenine (A) or guanine (G)).

As used herein, an emulsion “particle” refers to a oil droplet suspendedin the aqueous (continuous) phase of an oil-in-water emulsion. Theparticle further comprises a cationic liquid, and optionally additionalcomponents, such as a surfactant.

The term “polymer” refers to a molecule consisting of individualchemical moieties, which may be the same or different, that are joinedtogether. As used herein, the term “polymer” refers to individualchemical moieties that are joined end-to-end to form a linear molecule,as well as individual chemical moieties joined together in the form of abranched (e.g., a “multi-arm” or “star-shaped”) structure. Exemplarypolymers include, e.g., poloxamers. Poloxamers are nonionic triblockcopolymers having a central hydrophobic chain of polyoxypropylene(poly(propylene oxide)) flanked by two hydrophilic chains ofpolyoxyethylene (poly(ethylene oxide)).

As use herein, “saccharide” encompasses monosaccharides,oligosaccharides, or polysaccharides in straight chain or ring forms, ora combination thereof to form a saccharide chain. Oligosaccharides aresaccharides having two or more monosaccharide residues. Examples ofsaccharides include glucose, maltose, maltotriose, maltotetraose,sucrose and trehalose.

An emulsion is “stable” when the emulsion particles remain separatedwithout significant agglomeration or coalescence for at least one month,preferably at least two months, at 4° C. The average particle diameter(average number diameter) of a stable emulsion does not change by morethan 10% when the emulsion is stored at 4° C. for one month, orpreferably two months.

The term “surfactant” is a term of art and generally refers to anymolecule having both a hydrophilic group (e.g., a polar group), whichenergetically prefers solvation by water, and a hydrophobic group whichis not well solvated by water. The term “nonionic surfactant” is a knownterm in the art and generally refers to a surfactant molecule whosehydrophilic group (e.g., polar group) is not electrostatically charged.

The “Zeta potential” of an emulsion is determined by the electrophoreticmobility of the emulsion particles. The velocity of a particle in a unitelectric field is referred to as its electrophoretic mobility. Zetapotential is related to the electrophoretic mobility by the Henryequation:

$U_{E} = \frac{2ɛ\;{{zf}({ka})}}{3\eta}$where U_(E)=electrophoretic mobility, z=zeta potential, dielectricconstant, η=viscosity and f(ka)=Henry's function. Zeta potential istypically measured using an electrophoretic mobility apparatus, such asa Zetasizer Nano Z (Malvern Instruments Ltd, United Kingdom).

3. Cationic Oil-in-Water Emulsions

The cationic oil-in-water emulsions disclosed herein are generallydescribed in the manner that is conventional in the art, byconcentrations of components that are used to prepare the emulsions. Itis understood in the art that during the process of producing emulsions,including sterilization and other downstream processes, small amounts ofoil (e.g., squalene), cationic lipid (e.g., DOTAP), or other componentsmay be lost, and the actual concentrations of these components in thefinal product (e.g., a packaged, sterilized emulsion that is ready foradministration) might be slightly lower than starting amounts, sometimesby up to about 10%, by up to about 20%, by up to about 25%, or by up toabout 35%.

This invention generally relates to cationic oil-in-water emulsions thatcontain high concentrations of cationic lipids and a definedoil:cationic lipid ratio. The emulsions are particularly suitable fordelivering negatively charged molecules, such as an RNA molecule, to acell. The cationic lipid can interact with the negatively chargedmolecule, for example through electrostatic forces andhydrophobic/hydrophilic interactions, thereby anchoring the molecule tothe emulsion particles. The cationic emulsions described herein areuseful for delivering a negatively charged molecule, such as an RNAmolecule encoding an antigen or small interfering RNA to cells in vivo.For example, the cationic emulsions described herein provide advantagesfor delivering RNA molecules that encode one or more antigens, includingself-replicating RNAs, as vaccines.

The discrete phase (or dispersed phase) of the emulsion comprises an oiland a cationic lipid, wherein the cationic lipid facilitates dispersingthe oil in the aqueous (continuous) phase. One or more optionalcomponents may be present in the emulsion, such as surfactants (e.g.,nonionic surfactants) as described below.

The particles of the oil-in-water emulsions have an average diameter(i.e., average number diameter) of 1 micrometer or less. It isparticularly desirable that the average particle diameter of thecationic emulsions is about 180 nm or less, about 170 nm or less, about160 nm or less, about 150 nm or less, about 140 nm or less, about 130 nmor less, about 120 nm or less, about 110 nm or less, or about 100 nm orless; for example, from about 80 nm to 180 nm, from about 80 nm to 170nm, from about 80 nm to 160 nm, from about 80 nm to 150 nm, from about80 nm to 140 nm, from about 80 nm to 130 nm, from about 80 nm to 120 nm;from about 80 nm to 110 nm, or from about 80 nm to 100 nm. Particularlypreferred average particle diameter is about 100 nm, or from about 100nm to about 130 nm.

The size (average diameter) of the emulsion particles can be varied bychanging the ratio of surfactant to oil (increasing the ratio decreasesparticle size), operating pressure of homogenization (increasingoperating pressure of homogenization typically reduces particle size),temperature (increasing temperature decreases particle size), changingthe type of oil, inclusion of certain types of buffers in the aqueousphase, and other process parameters, as described in detail below. Insome cases, the size of the emulsion particles may affect theimmunogenicity of the RNA-emulsion complex, as exemplified herein.

The oil-in-water emulsions described herein are stable.

The particles of the emulsions described herein can be complexed with anegatively charged molecule. Prior to complexation with the negativelycharged molecule, the overall net charge of the particles (typicallymeasured as zeta-potential) should be positive (cationic). The overallnet charge of the particles may vary, depending on the type of thecationic lipid and the amount of the cationic lipid in the emulsion, theamount of oil in the emulsion (e.g., higher percentage of oil typicallyresults in less charge on the surface of the particles), and may also beaffected by any additional component (e.g., surfactant(s)) that ispresent in the emulsion. Preferably, the zeta-potential of thepre-complexation particles are no more than about 50 mV, no more thanabout 45 mV, no more than about 40 mV, no more than about 35 mV, no morethan about 30 mV, no more than about 25 mV, no more than about 20 mV;from about 5 mV to about 50 mV, from about 10 mV to about 50 mV, fromabout 10 mV to about 45 mV, from about 10 mV to about 40 mV, from about10 mV to about 35 mV, from about 10 mV to about 30 mV, from about 10 mVto about 25 mV, or from about 10 mV to about 20 mV. Zeta potential canbe affected by (i) pH of the emulsion, (ii) conductivity of the emulsion(e.g., salinity), and (iii) the concentration of the various componentsof the emulsion (polymer, non-ionic surfactants etc.). The Zetapotential of the cationic oil-in-water emulsions is measured using aMalvern Nanoseries Zetasizer (Westborough, Mass.). The sample is diluted1:100 in water (viscosity: 0.8872cp, RI: 1.330, Dielectric constant:78.5) and is added to a polystyrene latex capillary cell (Malvern,Westborough, Mass.). Zeta potential is measured at 25° C. with a 2minute equilibration time and analyzed using the Smoluchowski model(F(Ka) value=1.5). Data is reported in mV.

An exemplary cationic emulsion of the invention is referred herein as“CMF32.” The oil of CMF32 is squalene (at 4.3% w/v) and the cationiclipid is DOTAP (at 3.2 mg/mL). CMF32 also includes the surfactantsSPAN85 (sorbitan trioleate at 0.5% v/v) and Tween 80 (polysorbate 80;polyoxyethuylenesorbitan monooleate; at 0.5% v/v). Thus, emulsionparticles of CMF32 comprise squalene, SPAN85, Tween80, and DOTAP. RNAmolecules were shown to complex with CMF32 particles efficiently at 4:1,6:1, 8:1, 10:1, 12:1, and 14:1 N/P ratios. Other exemplary cationicemulsions include, e.g., the emulsions referred to herein as “CMF34”(4.3% w/v squalene, 0.5% Tween 80, 0.5% SPAN85, and 4.4 mg/mL DOTAP),“CMF35” (4.3% w/v squalene, 0.5% Tween 80, 0.5% SPAN85, 5.0 mg/mLDOTAP), and other emulsions described herein.

Certain exemplary cationic oil-in-water emulsions of the inventioncomprise DOTAP and squalene at concentrations of 2.1 mg/ml to 2.84 mg/ml(preferably 2.23 mg/ml to 2.71 mg/ml), and 30.92 mg/ml to 41.92 mg/ml(preferably 32.82 mg/ml to about 40.02 mg/ml), respectively, and furthercomprise equal amounts of SPAN85 and Tween80 (e.g., about 0.5% each).Other exemplary cationic oil-in-water emulsions of the inventioncomprise DOTAP and squalene at concentrations of 2.78 mg/ml to 3.76mg/ml (preferably 2.94 mg/ml to 3.6 mg/ml), and 18.6 mg/ml to 25.16mg/ml (preferably 19.69 mg/ml to about 24.07 mg/ml), respectively, andfurther comprise equal amounts of SPAN85 and Tween80 (e.g., about 0.5%each). Preferably, the particles of these emulsions have an averagediameter from 80 nm to 180 nm.

The individual components of the oil-in-water emulsions of the presentinvention are known in the art, although such compositions have not beencombined in the manner described herein. Accordingly, the individualcomponents, although described below both generally and in some-detailfor preferred embodiments, are well known in the art, and the terms usedherein, such as oil, surfactant, etc., are sufficiently well known toone skilled in the art without further description. In addition, whilepreferred ranges of the amount of the individual components of theemulsions are provided, the actual ratios of the components of aparticular emulsion may need to be adjusted so that emulsion particlesof desired size and physical property are properly formed. For example,if a particular amount of oil is used (e.g. 5% v/v oil), then, theamount of surfactant should be at level that is sufficient to dispersethe oil particle into the aqueous phase to form a stable emulsion. Theactual amount of surfactant required to disperse the oil into theaqueous phase depends on the type of surfactant and the type of oil usedfor the emulsion; and the amount of oil may also vary according to thedesired particle size (as this changes the surface area between the twophases). The actual amounts and the relative proportions of thecomponents of a desired emulsion can be readily determined by a skilledartisan.

A. Oil

The particles of the cationic oil-in-water emulsions comprise an oil.

The oil preferably is in the liquid phase at 1° C. or above, and isimmiscible to water.

Preferably, the oil is a metabolizable, non-toxic oil; more preferablyone of about 6 to about 30 carbon atoms including, but not limited to,alkanes, alkenes, alkynes, and their corresponding acids and alcohols,the ethers and esters thereof, and mixtures thereof. The oil may be anyvegetable oil, fish oil, animal oil or synthetically prepared oil thatcan be metabolized by the body of the subject to which the emulsion willbe administered, and is not toxic to the subject. The subject may be ananimal, typically a mammal, and preferably a human.

In certain embodiments, the oil is in liquid phase at 25° C. The oil isin liquid phase at 25° C., when it displays the properties of a fluid(as distinguished from solid and gas; and having a definite volume butno definite shape) when stored at 25° C. The emulsion, however, may bestored and used at any suitable temperature. Preferably, the oil is inliquid phase at 4° C.

The oil may be any long chain alkane, alkene or alkyne, or an acid oralcohol derivative thereof either as the free acid, its salt or an estersuch as a mono-, or di- or triester, such as the triglycerides andesters of 1,2-propanediol or similar poly-hydroxy alcohols. Alcohols maybe acylated employing a mono- or poly-functional acid, for exampleacetic acid, propanoic acid, citric acid or the like. Ethers derivedfrom long chain alcohols which are oils and meet the other criteria setforth herein may also be used.

The individual alkane, alkene or alkyne moiety and its acid or alcoholderivatives will generally have from about 6 to about 30 carbon atoms.The moiety may have a straight or branched chain structure. It may befully saturated or have one or more double or triple bonds. Where monoor poly ester- or ether-based oils are employed, the limitation of about6 to about 30 carbons applies to the individual fatty acid or fattyalcohol moieties, not the total carbon count.

Any suitable oils from an animal, fish or vegetable source may be used.Sources for vegetable oils include nuts, seeds and grains, and suitableoils peanut oil, soybean oil, coconut oil, and olive oil and the like.Other suitable seed oils include safflower oil, cottonseed oil,sunflower seed oil, sesame seed oil and the like. In the grain group,corn oil, and the oil of other cereal grains such as wheat, oats, rye,rice, teff, triticale and the like may also be used. The technology forobtaining vegetable oils is well developed and well known. Thecompositions of these and other similar oils may be found in, forexample, the Merck Index, and source materials on foods, nutrition andfood technology.

About six to about ten carbon fatty acid esters of glycerol and1,2-propanediol, while not occurring naturally in seed oils, may beprepared by hydrolysis, separation and esterification of the appropriatematerials starting from the nut and seed oils. These products arecommercially available under the name NEOBEES from PVO International,Inc., Chemical Specialties Division, 416 Division Street, Boongon, N.J.and others.

Animal oils and fats are often in solid phase at physiologicaltemperatures due to the fact that they exist as triglycerides and have ahigher degree of saturation than oils from fish or vegetables. However,fatty acids are obtainable from animal fats by partial or completetriglyceride saponification which provides the free fatty acids. Fatsand oils from mammalian milk are metabolizable and may therefore be usedin the practice of this invention. The procedures for separation,purification, saponification and other means necessary for obtainingpure oils from animal sources are well known in the art.

Most fish contain metabolizable oils which may be readily recovered. Forexample, cod liver oil, shark liver oils, and whale oil such asspermaceti exemplify several of the fish oils which may be used herein.A number of branched chain oils are synthesized biochemically in5-carbon isoprene units and are generally referred to as terpenoids.Squalene (2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene),a branched, unsaturated terpenoid, is particularly preferred herein. Amajor source of squalene is shark liver oil, although plant oils(primarily vegetable oils), including amaranth seed, rice bran, wheatgerm, and olive oils, are also suitable sources. Squalene can also beobtained from yeast or other suitable microbes. In some embodiments,Squalene is preferably obtained from non-animal sources, such as fromolives, olive oil or yeast. Squalane, the saturated analog to squalene,is also preferred. Fish oils, including squalene and squalane, arereadily available from commercial sources or may be obtained by methodsknown in the art.

In certain embodiments, the oil comprises an oil that is selected fromthe group consisting of: Castor oil, Coconut oil, Corn oil, Cottonseedoil, Evening primrose oil, Fish oil, Jojoba oil, Lard oil, Linseed oil,Olive oil, Peanut oil, Safflower oil, Sesame oil, Soybean oil, Squalene,Squalane, Sunflower oil and Wheatgerm oil. In exemplary embodiments, theoil comprises Squalene or Squalane.

The oil component of the emulsion may be present in an amount from about0.2% to about 10% (v/v). For example, the cationic oil-in-water emulsionmay comprise from about 0.2% to about 10% (v/v) oil, from about 0.2% toabout 9% (v/v) oil, from about 0.2% to about 8% (v/v) oil, from about0.2% to about 7% (v/v) oil, from about 0.2% to about 6% (v/v) oil, fromabout 0.2% to about 5% (v/v) oil, from about 0.3% to about 10% (v/v)oil, from about 0.4% to about 10% (v/v) oil, from about 0.5% to about10% (v/v) oil, from about 1% to about 10% (v/v) oil, from about 2% toabout 10% (v/v) oil, from about 3% to about 10% (v/v) oil, from about 4%to about 10% (v/v) oil, from about 5% to about 10% (v/v) oil, from about0.2% to about 10% (w/v) oil, from about 0.2% to about 9% (w/v) oil, fromabout 0.2% to about 8% (w/v) oil, from about 0.2% to about 7% (w/v) oil,from about 0.2% to about 6% (w/v) oil, from about 0.2% to about 5% (w/v)oil, from about 0.2% to about 4.3% (w/v) oil, from about 0.6% to about4% (w/v) oil, from about 0.7% to about 4% (w/v) oil, from about 0.8% toabout 4% (w/v) oil, from about 0.9% to about 4% (w/v) oil, from about1.0% to about 4% (w/v) oil, from about 0.6% to about 3.5% (w/v) oil,from about 0.6% to about 3% (w/v) oil, about 0.5% (v/v) oil, about 0.6%(v/v) oil, about 0.7% (v/v) oil, about 0.8% (v/v) oil, about 0.9% (v/v)oil, about 1% (v/v) oil, about 1.5% (v/v) oil, about 2% (v/v) oil, about2.5% (v/v) oil, about 3% (v/v) oil, about 3.5% (v/v) oil, about 4% (v/v)oil, about 5% (v/v) oil, about 10% (v/v) oil, about 0.5% (w/v) oil,about 1% (w/v) oil, about 1.5% (w/v) oil, about 2% (w/v) oil, about 2.5%(w/v) oil, about 3% (w/v) oil, about 3.5% (w/v) oil, about 4% (w/v) oil,about 4.3% (w/v) oil, about 5% (w/v) oil, about 5.5% (w/v) oil, about 6%(w/v) oil, about 6.5% (w/v) oil, about 7% (w/v) oil, about 7.5% (w/v)oil, or about 8% (w/v) oil.

The cationic oil-in-water emulsion may also comprise from about 0.2% toabout 8% (v/v) oil, for example, from 0.6% (w/v) to 4% (w/v), from about1% (w/v) to about 3.2% (w/v), about 1% (w/v), about 1.1% (w/v), about1.2% (w/v), about 1.3% (w/v), about 1.4% (w/v), about 1.5% (w/v), about1.6% (w/v), about 1.7% (w/v), about 1.8% (w/v), about 1.9% (w/v), about2.0% (w/v), about 2.1% (w/v), about 2.15% (w/v), about 2.2% (w/v), about2.3% (w/v), about 2.4% (w/v), about 2.5% (w/v), about 2.6% (w/v), about2.7% (w/v), about 2.8% (w/v), about 2.9% (w/v), 3.0% (w/v), about 3.1%(w/v), about 3.2% (w/v), about 3.3% (w/v), about 3.4% (w/v), about 3.5%(w/v), about 3.6% (w/v), about 3.7% (w/v), about 3.8% (w/v), about 3.9%(w/v), or about 4.0% (w/v) oil.

In an exemplary embodiment, the cationic oil-in-water emulsion comprisesabout 5% (v/v) oil. In another exemplary embodiment, the cationicoil-in-water emulsion comprises about 4.3% (w/v) squalene. In otherexemplary embodiments, the cationic oil-in-water emulsion comprises from0.6% (w/v) to 4% (w/v) squalene, for example, from about 1% (w/v) toabout 3.2% (w/v) squalene, such as 1.08% (w/v), 2.15% (w/v), or 3.23%(w/v) squalene, as shown in the Examples.

As noted above, the percentage of oil described above is determinedbased on the initial amount of the oil that is used to prepare theemulsions. It is understood in the art that the actual concentration ofthe oil in the final product (e.g., a packaged, sterilized emulsion thatis ready for administration) might be slightly lower, sometimes by up toabout 10%, by up to about 20%, by up to about 25%, or by up to about35%.

B. Cationic Lipids

The emulsion particles described herein comprise a cationic lipid, whichcan interact with the negatively charged molecule thereby anchoring themolecule to the emulsion particles.

Any suitable cationic lipid may be used. Generally, the cationic lipidcontains a nitrogen atom that is positively charged under physiologicalconditions. The head group of the cationic lipid can comprise a tertiaryamine or, preferably, a quaternary amine Certain suitable cationiclipids comprise two saturated or unsaturated fatty acid chains (e.g.,side chains having from about 10 to about 30 carbon atoms).

The cationic lipid can have a positive charge at about 12 pH, about 11pH, about 10 pH, about 9 pH, about 8 pH, about 7 pH, or about 6 pH.

Suitable cationic lipids include, benzalkonium chloride (BAK),benzethonium chloride, cetrimide (which containstetradecyltrimethylammonium bromide and possibly small amounts ofdodecyltrimethylammonium bromide and hexadecyltrimethyl ammoniumbromide), cetylpyridinium chloride (CPC), cetyl trimethylammoniumchloride (CTAC), primary amines, secondary amines, tertiary amines,including but not limited to N,N′,N′-polyoxyethylene(10)-N-tallow-1,3-diaminopropane, other quaternary amine salts,including but not limited to dodecyltrimethylammonium bromide,hexadecyltrimethyl-ammonium bromide, mixed alkyl-trimethyl-ammoniumbromide, benzyldimethyldodecylammonium chloride,benzyldimethylhexadecyl-ammonium chloride, benzyltrimethylammoniummethoxide, cetyldimethylethylammonium bromide, dimethyldioctadecylammonium bromide (DDAB), methylbenzethonium chloride, decamethoniumchloride, methyl mixed trialkyl ammonium chloride, methyltrioctylammonium chloride), N,N-dimethyl-N-[2(2-methyl-4-(1,1,3,3tetramethylbutyl)-phenoxyl-ethoxy)ethyl]-benzenemetha-naminiumchloride (DEBDA), dialkyldimetylammonium salts,[1-(2,3-dioleyloxy)-propyl]-N,N,N,trimethylammonium chloride,1,2-diacyl-3-(trimethylammonio) propane (acyl group=dimyristoyl,dipalmitoyl, distearoyl, dioleoyl), 1,2-diacyl-3(dimethylammonio)propane (acyl group=dimyristoyl, dipalmitoyl,distearoyl, dioleoyl),1,2-dioleoyl-3-(4′-trimethyl-ammonio)butanoyl-sn-glycerol, 1,2-dioleoyl3-succinyl-sn-glycerol choline ester, cholesteryl (4′-trimethylammonio)butanoate), N-alkyl pyridinium salts (e.g. cetylpyridinium bromide andcetylpyridinium chloride), N-alkylpiperidinium salts, dicationicbolaform electrolytes (C₁₂Me₆; C₁₂Bu₆),dialkylglycetylphosphorylcholine, lysolecithin, L-αdioleoylphosphatidylethanolamine, cholesterol hemisuccinate cholineester, lipopolyamines, including but not limited todioctadecylamidoglycylspermine (DOGS), dipalmitoylphosphatidylethanol-amidospermine (DPPES), lipopoly-L (or D)-lysine(LPLL, LPDL), poly (L (or D)-lysine conjugated toN-glutarylphosphatidylethanolamine, didodecyl glutamate ester withpendant amino group (C₁₂GluPhC_(n)N⁺), ditetradecyl glutamate ester withpendant amino group (C₁₄GluC_(n)N⁺), cationic derivatives ofcholesterol, including but not limited to cholesteryl-3β-oxysuccinamidoethylenetrimethylammonium salt, cholesteryl-3β-oxysuccinamidoethylenedimethylamine, cholesteryl-3β-carboxyamidoethylenetrimethylammonium salt, cholesteryl-3β-carboxyamidoethylenedimethylamine, and3γ-[N—(N′,N-dimethylaminoetanecarbomoyl]cholesterol) (DC-Cholesterol),1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP),dimethyldioctadecylammonium (DDA),1,2-Dimyristoyl-3-TrimethylAmmoniumPropane (DMTAP),dipalmitoyl(C_(16:0))trimethyl ammonium propane (DPTAP),distearoyltrimethylammonium propane (DSTAP), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA),N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC),1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC),1,2-dioleoyl-3-dimethylammonium-propane (DODAP),1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), and combinationthereof.

In preferred embodiments, the cationic lipid is selected from the groupconsisting of 1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP),1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC),N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), and N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). Incertain embodiments, the cationic lipid is not DC-Cholesterol.

Preferably, the cationic lipid selected for the emulsion is soluble inthe oil that is selected for the emulsion. This permits high cationiclipid concentrations to be achieved in the emulsion, by directlydissolving the lipid in the oil prior to dispersion in the mobile phase.It is within the knowledge in the art to determine whether a particularlipid is soluble in the oil and choose an appropriate oil and lipidcombination accordingly. For example, solubility can be predicted basedon the structures of the lipid and oil (e.g., the solubility of a lipidmay be determined by the structure of its tail). For example, lipidshaving one or two unsaturated fatty acid chains (e.g., oleoyl tails, orlinolyl tails), such as DOTAP, DOEPC, DODAC, DOTMA, are soluble insqualene or squalane. Alternatively, solubility can be determinedaccording to the quantity of the lipid that dissolves in a givenquantity of the oil to form a saturated solution. Such methods are knownin the art. The solubility of exemplary saturated or unsaturated fattyacids in squalene is also provided in the Examples. Preferably, thesaturation concentration of the lipid in the oil is at least about 1mg/ml, at least about 5 mg/ml, at least about 10 mg/ml, at least about25 mg/ml, at least about 50 mg/ml or at least about 100 mg/ml.

Preferably, the concentration of cationic lipid in the emulsion beforethe negatively charged molecule is complexed is at least about 1.25 mM,at least about 1.5 mM, at least about 1.75 mM, at least about 2.0 mM, atleast about 2.25 mM, at least about 2.5 mM, at least about 2.75 mM, atleast about 3.0 mM, at least about 3.25 mM, at least about 3.5 mM, atleast about 3.75 mM, at least about 4.0 mM, at least about 4.25 mM, atleast about 4.5 mM, at least about 4.75 mM, at least about 5.0 mM, atleast about 5.25 mM, at least about 5.5 mM, at least about 5.75 mM, atleast about 6 mM, at least about 6.25 mM, at least about 6.5 mM, atleast about 6.75 mM, at least about 7 mM, at least about 7.25 mM, atleast about 7.5 mM, at least about 7.75 mM, at least about 8 mM, atleast about 8.25 mM, at least about 8.5 mM, at least about 8.75 mM, atleast about 9 mM, at least about 9.25 mM, at least about 9.5 mM, atleast about 9.75 mM, or at least about 10 mM.

In certain embodiments, the cationic lipid is DOTAP. The cationicoil-in-water emulsion may comprise from about 0.8 mg/ml to about 10mg/ml DOTAP. For example, the cationic oil-in-water emulsion maycomprise DOTAP at from about 1.7 mg/ml to about 10 mg/ml, from about 1.8mg/ml to about 10 mg/ml, from about 2.0 mg/ml to about 10 mg/ml, fromabout 2.2 mg/ml to about 10 mg/ml, from about 2.4 mg/ml to about 10mg/ml, from about 2.6 mg/ml to about 10 mg/ml, from about 2.8 mg/ml toabout 10 mg/ml, from about 3.0 mg/ml to about 10 mg/ml, from about 3.2mg/ml to about 10 mg/ml, from about 3.4 mg/ml to about 10 mg/ml, fromabout 3.6 mg/ml to about 10 mg/ml, from about 4.0 mg/ml to about 10mg/ml, from about 4.4 mg/ml to about 10 mg/ml, from about 4.8 mg/ml toabout 10 mg/ml, from about 5 mg/ml to about 10 mg/ml, from about 1.7mg/ml to about 5 mg/ml, from about 1.8 mg/ml to about 5 mg/ml, fromabout 1.8 mg/ml to about 6 mg/ml, from about 1.8 mg/ml to about 7 mg/ml,from about 1.8 mg/ml to about 8 mg/ml, from about 1.8 mg/ml to about 9mg/ml, about 1.7 mg/ml, about 1.8 mg/ml, about 2.0 mg/ml, about 2.2mg/ml, about 2.4 mg/ml, about 2.6 mg/ml, about 2.8 mg/ml, about 3.0mg/ml, about 3.2 mg/ml, about 3.4 mg/ml, about 3.6 mg/ml, about 3.8mg/ml, about 4.0 mg/ml, about 4.2 mg/ml, about 4.4 mg/ml, about 4.6mg/ml, about 4.8 mg/ml, about 5.0 mg/ml, about 5.2 mg/ml, about 5.5mg/ml, about 6.0 mg/ml, at least about 0.8 mg/ml, at least about 0.85mg/ml, at least about 0.9 mg/ml, at least about 1.0 mg/ml, at leastabout 1.1 mg/ml, at least about 1.2 mg/ml, at least about 1.3 mg/ml, atleast about 1.4 mg/ml, at least about 1.5 mg/ml, at least about 1.6mg/ml, at least about 1.7 mg/ml, etc.

In an exemplary embodiment, the cationic oil-in-water emulsion comprisesfrom about 1.8 mg/ml to about 5.0 mg/ml DOTAP.

In certain embodiments, the cationic lipid is DOEPC. The cationicoil-in-water emulsion may comprise from about 0.8 mg/ml to about 10mg/ml DOEPC. For example, the cationic oil-in-water emulsion maycomprise DOEPC at from about 1.7 mg/ml to about 10 mg/ml, from about 1.8mg/ml to about 10 mg/ml, from about 2.0 mg/ml to about 10 mg/ml, fromabout 2.2 mg/ml to about 10 mg/ml, from about 2.4 mg/ml to about 10mg/ml, from about 2.6 mg/ml to about 10 mg/ml, from about 2.8 mg/ml toabout 10 mg/ml, from about 3.0 mg/ml to about 10 mg/ml, from about 3.2mg/ml to about 10 mg/ml, from about 3.4 mg/ml to about 10 mg/ml, fromabout 3.6 mg/ml to about 10 mg/ml, from about 4.0 mg/ml to about 10mg/ml, from about 4.4 mg/ml to about 10 mg/ml, from about 4.8 mg/ml toabout 10 mg/ml, from about 5 mg/ml to about 10 mg/ml, from about 1.7mg/ml to about 5 mg/ml, from about 1.8 mg/ml to about 5 mg/ml, fromabout 1.8 mg/ml to about 6 mg/ml, from about 1.8 mg/ml to about 7 mg/ml,from about 1.8 mg/ml to about 8 mg/ml, from about 1.8 mg/ml to about 9mg/ml, about 1.7 mg/ml, about 1.8 mg/ml, about 2.0 mg/ml, about 2.2mg/ml, about 2.4 mg/ml, about 2.6 mg/ml, about 2.8 mg/ml, about 3.0mg/ml, about 3.2 mg/ml, about 3.4 mg/ml, about 3.6 mg/ml, about 3.8mg/ml, about 4.0 mg/ml, about 4.2 mg/ml, about 4.4 mg/ml, about 4.6mg/ml, about 4.8 mg/ml, about 5.0 mg/ml, about 5.2 mg/ml, about 5.5mg/ml, about 6.0 mg/ml, at least about 0.8 mg/ml, at least about 0.85mg/ml, at least about 0.9 mg/ml, at least about 1.0 mg/ml, at leastabout 1.1 mg/ml, at least about 1.2 mg/ml, at least about 1.3 mg/ml, atleast about 1.4 mg/ml, at least about 1.5 mg/ml, at least about 1.6mg/ml, at least about 1.7 mg/ml, etc.

In certain embodiments, the cationic lipid is DODAC. The cationicoil-in-water emulsion may comprise from about 0.8 mg/ml to about 10mg/ml DODAC. For example, the cationic oil-in-water emulsion maycomprise DODAC at from about 1.7 mg/ml to about 10 mg/ml, from about 1.8mg/ml to about 10 mg/ml, from about 2.0 mg/ml to about 10 mg/ml, fromabout 2.2 mg/ml to about 10 mg/ml, from about 2.4 mg/ml to about 10mg/ml, from about 2.6 mg/ml to about 10 mg/ml, from about 2.8 mg/ml toabout 10 mg/ml, from about 3.0 mg/ml to about 10 mg/ml, from about 3.2mg/ml to about 10 mg/ml, from about 3.4 mg/ml to about 10 mg/ml, fromabout 3.6 mg/ml to about 10 mg/ml, from about 4.0 mg/ml to about 10mg/ml, from about 4.4 mg/ml to about 10 mg/ml, from about 4.8 mg/ml toabout 10 mg/ml, from about 5 mg/ml to about 10 mg/ml, from about 1.7mg/ml to about 5 mg/ml, from about 1.8 mg/ml to about 5 mg/ml, fromabout 1.8 mg/ml to about 6 mg/ml, from about 1.8 mg/ml to about 7 mg/ml,from about 1.8 mg/ml to about 8 mg/ml, from about 1.8 mg/ml to about 9mg/ml, about 1.7 mg/ml, about 1.8 mg/ml, about 2.0 mg/ml, about 2.2mg/ml, about 2.4 mg/ml, about 2.6 mg/ml, about 2.8 mg/ml, about 3.0mg/ml, about 3.2 mg/ml, about 3.4 mg/ml, about 3.6 mg/ml, about 3.8mg/ml, about 4.0 mg/ml, about 4.2 mg/ml, about 4.4 mg/ml, about 4.6mg/ml, about 4.8 mg/ml, about 5.0 mg/ml, about 5.2 mg/ml, about 5.5mg/ml, about 6.0 mg/ml, at least about 0.8 mg/ml, at least about 0.85mg/ml, at least about 0.9 mg/ml, at least about 1.0 mg/ml, at leastabout 1.1 mg/ml, at least about 1.2 mg/ml, at least about 1.3 mg/ml, atleast about 1.4 mg/ml, at least about 1.5 mg/ml, at least about 1.6mg/ml, at least about 1.7 mg/ml, etc.

In certain embodiments, the cationic lipid is DOTMA. The cationicoil-in-water emulsion may comprise from about 0.8 mg/ml to about 10mg/ml DOTMA. For example, the cationic oil-in-water emulsion maycomprise DOTMA at from about 1.7 mg/ml to about 10 mg/ml, from about 1.8mg/ml to about 10 mg/ml, from about 2.0 mg/ml to about 10 mg/ml, fromabout 2.2 mg/ml to about 10 mg/ml, from about 2.4 mg/ml to about 10mg/ml, from about 2.6 mg/ml to about 10 mg/ml, from about 2.8 mg/ml toabout 10 mg/ml, from about 3.0 mg/ml to about 10 mg/ml, from about 3.2mg/ml to about 10 mg/ml, from about 3.4 mg/ml to about 10 mg/ml, fromabout 3.6 mg/ml to about 10 mg/ml, from about 4.0 mg/ml to about 10mg/ml, from about 4.4 mg/ml to about 10 mg/ml, from about 4.8 mg/ml toabout 10 mg/ml, from about 5 mg/ml to about 10 mg/ml, from about 1.7mg/ml to about 5 mg/ml, from about 1.8 mg/ml to about 5 mg/ml, fromabout 1.8 mg/ml to about 6 mg/ml, from about 1.8 mg/ml to about 7 mg/ml,from about 1.8 mg/ml to about 8 mg/ml, from about 1.8 mg/ml to about 9mg/ml, about 1.7 mg/ml, about 1.8 mg/ml, about 2.0 mg/ml, about 2.2mg/ml, about 2.4 mg/ml, about 2.6 mg/ml, about 2.8 mg/ml, about 3.0mg/ml, about 3.2 mg/ml, about 3.4 mg/ml, about 3.6 mg/ml, about 3.8mg/ml, about 4.0 mg/ml, about 4.2 mg/ml, about 4.4 mg/ml, about 4.6mg/ml, about 4.8 mg/ml, about 5.0 mg/ml, about 5.2 mg/ml, about 5.5mg/ml, about 6.0 mg/ml, at least about 0.8 mg/ml, at least about 0.85mg/ml, at least about 0.9 mg/ml, at least about 1.0 mg/ml, at leastabout 1.1 mg/ml, at least about 1.2 mg/ml, at least about 1.3 mg/ml, atleast about 1.4 mg/ml, at least about 1.5 mg/ml, at least about 1.6mg/ml, at least about 1.7 mg/ml, etc.

As noted above, the concentration of a lipid described above isdetermined based on the initial amount of the lipid that is used toprepare the emulsions. It is understood in the art that the actualconcentration of the oil in the final product (e.g., a packaged,sterilized emulsion that is ready for administration) might be slightlylower, sometimes by up to about 10%, by up to about 20%, by up to about25%, or by up to about 35%.

C. Oil to Lipid Ratio

The cationic oil-in-water emulsions of the invention have a definedoil:lipid ratio. For example, the ratio of oil:lipid (mole:mole) of theemulsion may be at least about 8:1 (mole:mole), at least about 8.5:1(mole:mole), at least about 9:1 (mole:mole), at least about 9.5:1(mole:mole), at least about 10:1 (mole:mole), at least about 10.5:1(mole:mole), at least about 11:1 (mole:mole), at least about 11.5:1(mole:mole), at least about 12:1 (mole:mole), at least about 12.5:1(mole:mole), at least about 13:1 (mole:mole), at least about 13.5:1(mole:mole), at least about 14:1 (mole:mole), at least about 14.5:1(mole:mole), at least about 15:1 (mole:mole), at least about 15.5:1(mole:mole), at least about 16:1 (mole:mole), at least about 16.5:1(mole:mole), at least about 17:1 (mole:mole), from about 8:1 (mole:mole)to about 50:1 (mole:mole), from about 9:1 (mole:mole) to about 50:1(mole:mole), from about 10:1 (mole:mole) to about 50:1 (mole:mole), fromabout 8:1 (mole:mole) to about 49:1 (mole:mole), from about 8:1(mole:mole) to about 48:1 (mole:mole), from about 8:1 (mole:mole) toabout 47:1 (mole:mole), from about 8:1 (mole:mole) to about 46:1(mole:mole), from about 8:1 (mole:mole) to about 45:1 (mole:mole), fromabout 8:1 (mole:mole) to about 44:1 (mole:mole), from about 8:1(mole:mole) to about 43:1 (mole:mole), from about 8:1 (mole:mole) toabout 42:1 (mole:mole), from about 8:1 (mole:mole) to about 41:1(mole:mole), from about 9:1 (mole:mole) to about 43:1 (mole:mole), fromabout 10:1 (mole:mole) to about 43:1 (mole:mole), from about 11:1(mole:mole) to about 43:1 (mole:mole), from about 12:1 (mole:mole) toabout 43:1 (mole:mole), from about 13:1 (mole:mole) to about 43:1(mole:mole), from about 14:1 (mole:mole) to about 43:1 (mole:mole), fromabout 15:1 (mole:mole) to about 43:1 (mole:mole), from about 16:1(mole:mole) to about 43:1 (mole:mole), from about 17:1 (mole:mole) toabout 43:1 (mole:mole), etc.

If desired, the ratio of oil:lipid (mole:mole) of the emulsion may be atleast about 4:1 (mole:mole), at least about 4.2:1 (mole:mole), at leastabout 4.5:1 (mole:mole), at least about 5:1 (mole:mole), at least about5.5:1 (mole:mole), at least about 6:1 (mole:mole), at least about 6.5:1(mole:mole), 7:1 (mole:mole), at least about 7.5:1 (mole:mole), fromabout 4:1 (mole:mole) to about 50:1 (mole:mole), from about 5:1(mole:mole) to about 50:1 (mole:mole), from about 6:1 (mole:mole) toabout 50:1 (mole:mole), from about 7:1 (mole:mole) to about 50:1(mole:mole), from about 4:1 (mole:mole) to about 49:1 (mole:mole), fromabout 4:1 (mole:mole) to about 48:1 (mole:mole), from about 4:1(mole:mole) to about 47:1 (mole:mole), from about 4:1 (mole:mole) toabout 46:1 (mole:mole), from about 4:1 (mole:mole) to about 45:1(mole:mole), from about 4:1 (mole:mole) to about 44:1 (mole:mole), fromabout 4:1 (mole:mole) to about 43:1 (mole:mole), from about 4:1(mole:mole) to about 42:1 (mole:mole), from about 4:1 (mole:mole) toabout 41:1 (mole:mole), from about 5:1 (mole:mole) to about 43:1(mole:mole), from about 6:1 (mole:mole) to about 43:1 (mole:mole), fromabout 7:1 (mole:mole) to about 43:1 (mole:mole), etc.

Sometimes, there may be a need to strike a balance between the desire toincrease the concentration of a cationic lipid (thereby increasing theamount of nucleic acid molecules loaded to the emulsion particle), andtoxicity or tolerability of the lipid when administered in vivo. Forexample, it has been reported that high doses of DOTAP can have toxiceffects. See, e.g., Lappalainen et al., Pharm. Res., vol. 11(8):1127-31(1994). The optimal range of lipid dose in a particular emulsion can bedetermined in accordance with the knowledge of a skilled clinician.

If the oil comprises a mixture of molecules, the molar concentration ofthe oil can be calculated based on the average molecular weight of theoil. For example, the average molecular weight of soybean oil (292.2)can be calculated according to the average fatty acid distribution (12%weight percentage of palmitic acid; 52% weight percentage of linolenicacid; etc), and the molecular weight of each component.

C. Additional Components

The cationic oil-in-water emulsions described herein may furthercomprise additional components. For example, the emulsions may comprisecomponents that can promote particle formation, improve the complexationbetween the negatively charge molecules and the cationic particles, orincrease the stability of the negatively charge molecule (e.g., toprevent degradation of an RNA molecule). If desired, the cationicoil-in-water emulsion can contain an antioxidant, such as citrate,ascorbate or salts thereof.

Surfactants

In certain embodiments, the cationic oil-in-water emulsion as describedherein further comprises a surfactant.

A substantial number of surfactants have been used in the pharmaceuticalsciences. These include naturally derived materials such as gums fromtrees, vegetable protein, sugar-based polymers such as alginates, andthe like. Certain oxypolymers or polymers having a hydroxide or otherhydrophilic substituent on the carbon backbone have surfactant activity,for example, povidone, polyvinyl alcohol, and glycol ether-based mono-and poly-functional compounds. Ionic or nonionic detergents and longchain fatty-acid-derived compounds can also be used in this invention.

Specific examples of suitable surfactants include the following:

1. Water-soluble soaps, such as the sodium, potassium, ammonium andalkanol-ammonium salts of higher fatty acids (C₁₀-C₂₂), in particularsodium and potassium tallow and coconut soaps.

2. Anionic synthetic non-soap surfactants, which can be represented bythe water-soluble salts of organic sulfuric acid reaction productshaving in their molecular structure an alkyl radical containing fromabout 8 to 22 carbon atoms and a radical selected from the groupconsisting of sulfonic acid and sulfuric acid ester radicals. Examplesof these are the sodium or potassium alkyl sulfates, derived from tallowor coconut oil; sodium or potassium alkyl benzene sulfonates; sodiumalkyl glyceryl ether sulfonates; sodium coconut oil fatty acidmonoglyceride sulfonates and sulfates; sodium or potassium salts ofsulfuric acid esters of the reaction product of one mole of a higherfatty alcohol and about 1 to 6 moles of ethylene oxide; sodium orpotassium alkyl phenol ethylene oxide ether sulfonates, with 1 to 10units of ethylene oxide per molecule and in which the alkyl radicalscontain from 8 to 12 carbon atoms; the reaction product of fatty acidsesterified with isethionic acid and neutralized with sodium hydroxide;sodium or potassium salts of fatty acid amide of a methyl tauride; andsodium and potassium salts of SO₃-sulfonated C₁₀-C₂₄ α-olefins.

3. Nonionic synthetic surfactants made by the condensation of alkyleneoxide groups with an organic hydrophobic compound. Typical hydrophobicgroups include condensation products of propylene oxide with propyleneglycol, alkyl phenols, condensation product of propylene oxide andethylene diamine, aliphatic alcohols having 8 to 22 carbon atoms, andamides of fatty acids.

4. Nonionic surfactants, such as amine oxides, phosphine oxides andsulfoxides, having semipolar characteristics. Specific examples of longchain tertiary amine oxides include dimethyldodecylamine oxide andbis-(2-hydroxyethyl) dodecylamine Specific examples of phosphine oxidesare found in U.S. Pat. No. 3,304,263, issued Feb. 14, 1967, and includedimethyldodecylphosphine oxide and dimethyl-(2hydroxydodecyl) phosphineoxide.

5. Long chain sulfoxides, including those corresponding to the formulaR¹—SO—R² wherein R¹ and R² are substituted or unsubstituted alkylradicals, the former containing from about 10 to about 28 carbon atoms,whereas R² contains from 1 to 3 carbon atoms. Specific examples of thesesulfoxides include dodecyl methyl sulfoxide and 3-hydroxy tridecylmethyl sulfoxide.

6. Ampholytic synthetic surfactants, such as sodium3-dodecylaminopropionate and sodium 3-dodecylaminopropane sulfonate.

7. Zwitterionic synthetic surfactants, such as3-(N,N-dimethyl-N-hexadecylammonio)propane-1-sulfonate and3-(N,N-dimethyl-N-hexadecylammonio)-2-hydroxy propane-1-sulfonate.

Additionally, all of the following types of surfactants can be used in acomposition of the present invention: (a) soaps (i.e., alkali salts) offatty acids, rosin acids, and tall oil; (b) alkyl arene sulfonates; (c)alkyl sulfates, including surfactants with both branched-chain andstraight-chain hydrophobic groups, as well as primary and secondarysulfate groups; (d) sulfates and sulfonates containing an intermediatelinkage between the hydrophobic and hydrophilic groups, such as thefatty acylated methyl taurides and the sulfated fatty monoglycerides;(e) long-chain acid esters of polyethylene glycol, especially the talloil esters; (f) polyethylene glycol ethers of alkylphenols; (g)polyethylene glycol ethers of long-chain alcohols and mercaptans; and(h) fatty acyl diethanol amides. Since surfactants can be classified inmore than one manner, a number of classes of surfactants set forth inthis paragraph overlap with previously described surfactant classes.

There are a number of surfactants specifically designed for and commonlyused in biological situations. Such surfactants are divided into fourbasic types: anionic, cationic, zwitterionic (amphoteric), and nonionic.Exemplary anionic surfactants include, e.g., perfluorooctanoate (PFOA orPFO), perfluorooctanesulfonate (PFOS), alkyl sulfate salts such assodium dodecyl sulfate (SDS) or ammonium lauryl sulfate, sodium laurethsulfate (also known as sodium lauryl ether sulfate, SLES), alkyl benzenesulfonate, and fatty acid salts. Exemplary cationic surfactants include,e.g., alkyltrimethylammonium salts such as cetyl trimethylammoniumbromide (CTAB, or hexadecyl trimethyl ammonium bromide), cetylpyridiniumchloride (CPC), polyethoxylated tallow amine (POEA), benzalkoniumchloride (BAC), benzethonium chloride (BZT). Exemplary zwitterionic(amphoteric) surfactants include, e.g., dodecyl betaine, cocamidopropylbetaine, and coco ampho glycinate. Exemplary nonionic surfactantsinclude, e.g., alkyl poly(ethylene oxide), alkylphenol poly(ethyleneoxide), copolymers of poly(ethylene oxide) and poly(propylene oxide)(commercially called poloxamers or poloxamines), Aayl polyglucosides(e.g., octyl glucoside or decyl maltoside), fatty alcohols (e.g., cetylalcohol or oleyl alcohol), cocamide MEA, cocamide DEA, Pluronic® F-68(polyoxyethylene-polyoxypropylene block copolymer), and polysorbates,such as Tween 20 (polysorbate 20), Tween 80 (polysorbate 80;polyoxyethuylenesorbitan monooleate), dodecyl dimethylamine oxide, andvitamin E tocopherol propylene glycol succinate (Vitamin E TPGS).

A particularly useful group of surfactants are the sorbitan-basednon-ionic surfactants. These surfactants are prepared by dehydration ofsorbitol to give 1,4-sorbitan which is then reacted with one or moreequivalents of a fatty acid. The fatty-acid-substituted moiety may befurther reacted with ethylene oxide to give a second group ofsurfactants.

The fatty-acid-substituted sorbitan surfactants are made by reacting1,4-sorbitan with a fatty acid such as lauric acid, palmitic acid,stearic acid, oleic acid, or a similar long chain fatty acid to give the1,4-sorbitan mono-ester, 1,4-sorbitan sesquiester or 1,4-sorbitantriester. The common names for these surfactants include, for example,sorbitan monolaurate, sorbitan monopalmitate, sorbitan monoestearate,sorbitan monooleate, sorbitan sesquioleate, and sorbitan trioleate.These surfactants are commercially available under the name SPAN® orARLACEL®, usually with a letter or number designation whichdistinguishes between the various mono, di- and triester substitutedsorbitans.

SPAN® and ARLACEL® surfactants are hydrophilic and are generally solubleor dispersible in oil. They are also soluble in most organic solvents.In water they are generally insoluble but dispersible. Generally thesesurfactants will have a hydrophilic-lipophilic balance (HLB) numberbetween 1.8 to 8.6. Such surfactants can be readily made by means knownin the art or are commercially available.

A related group of surfactants comprises olyoxyethylene sorbitanmonoesters and olyoxyethylene sorbitan triesters. These materials areprepared by addition of ethylene oxide to a 1,4-sorbitan monester ortriester. The addition of polyoxyethylene converts the lipophilicsorbitan mono- or triester surfactant to a hydrophilic surfactantgenerally soluble or dispersible in water and soluble to varying degreesin organic liquids.

These materials, commercially available under the mark TWEEN®, areuseful for preparing oil-in-water emulsions and dispersions, or for thesolubilization of oils and making anhydrous ointments water-soluble orwashable. The TWEEN® surfactants may be combined with a related sorbitanmonester or triester surfactants to promote emulsion stability. TWEEN®surfactants generally have a HLB value falling between 9.6 to 16.7.TWEEN® surfactants are commercially available.

A third group of non-ionic surfactants which could be used alone or inconjunction with SPANS, ARLACEL® and TWEEN® surfactants are thepolyoxyethylene fatty acids made by the reaction of ethylene oxide witha long-chain fatty acid. The most commonly available surfactant of thistype is solid under the name MYRJ® and is a polyoxyethylene derivativeof stearic acid. MYRJ® surfactants are hydrophilic and soluble ordispersible in water like TWEEN® surfactants. The MYRJ® surfactants maybe blended with TWEEN® surfactants or with TWEEN®/SPAN® or ARLACEL®surfactant mixtures for use in forming emulsions. MYRJ® surfactants canbe made by methods known in the art or are available commercially.

A fourth group of polyoxyethylene based non-ionic surfactants are thepolyoxyethylene fatty acid ethers derived from lauryl, acetyl, stearyland oleyl alcohols. These materials are prepared as above by addition ofethylene oxide to a fatty alcohol. The commercial name for thesesurfactants is BRIJ®. BRIJ® surfactants may be hydrophilic or lipophilicdepending on the size of the polyoxyethylene moiety in the surfactant.While the preparation of these compounds is available from the art, theyare also readily available from commercial sources.

Other non-ionic surfactants which could potentially be used are, forexample, polyoxyethylene, polyol fatty acid esters, polyoxyethyleneether, polyoxypropylene fatty ethers, bee's wax derivatives containingpolyoxyethylene, polyoxyethylene lanolin derivative, polyoxyethylenefatty glycerides, glycerol fatty acid esters or other polyoxyethyleneacid alcohol or ether derivatives of long-chain fatty acids of 12-22carbon atoms.

As the emulsions and formulations of the invention are intended to bemulti-phase systems, it is preferable to choose an emulsion-formingnon-ionic surfactant which has an HLB value in the range of about 7 to16. This value may be obtained through the use of a single non-ionicsurfactant such as a TWEEN® surfactant or may be achieved by the use ofa blend of surfactants such as with a sorbitan mono, di- or triesterbased surfactant; a sorbitan ester polyoxyethylene fatty acid; asorbitan ester in combination with a polyoxyethylene lanolin derivedsurfactant; a sorbitan ester surfactant in combination with a high HLBpolyoxyethylene fatty ether surfactant; or a polyethylene fatty ethersurfactant or polyoxyethylene sorbitan fatty acid.

In certain embodiments, the emulsion comprises a single non-ionicsurfactant, most particularly a TWEEN® surfactant, as the emulsionstabilizing non-ionic surfactant. In an exemplary embodiment, theemulsion comprises TWEEN® 80, otherwise known as polysorbate 80 orpolyoxyethylene 20 sorbitan monooleate. In other embodiments, theemulsion comprises two or more non-ionic surfactants, in particular aTWEEN® surfactant and a SPAN® surfactant. In an exemplary embodiment,the emulsion comprises TWEEN® 80 and SPAN®85.

The oil-in-water emulsions can contain from about 0.01% to about 2.5%surfactant (w/v), about 0.01% to about 2% surfactant, 0.01% to about1.5% surfactant, 0.01% to about 1% surfactant, 0.01% to about 0.5%surfactant, 0.05% to about 0.5% surfactant, 0.08% to about 0.5%surfactant, about 0.08% surfactant, about 0.1% surfactant, about 0.2%surfactant, about 0.3% surfactant, about 0.4% surfactant, about 0.5%surfactant, about 0.6% surfactant, about 0.7% surfactant, about 0.8%surfactant, about 0.9% surfactant, or about 1% surfactant.

Alternatively or in addition, the oil-in-water emulsions can contain0.05% to about 1%, 0.05% to about 0.9%, 0.05% to about 0.8%, 0.05% toabout 0.7%, 0.05% to about 0.6%, 0.05% to about 0.5%, about 0.08%, about0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about0.7%, about 0.8%, about 0.9%, or about 1% (w/v) Tween 80 (polysorbate80; polyoxyethuylenesorbitan monooleate).

In an exemplary embodiment, the oil-in-water emulsion contains 0.08%(w/v) Tween 80 (polysorbate 80; polyoxyethuylenesorbitan monooleate).

Alternatively or in addition, the oil-in-water emulsions can contain0.05% to about 1%, 0.05% to about 0.9%, 0.05% to about 0.8%, 0.05% toabout 0.7%, 0.05% to about 0.6%, 0.05% to about 0.5%, about 0.08%, about0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about0.7%, about 0.8%, about 0.9%, or about 1% (w/v) SPAN85 (sorbitantrioleate).

The oil-in-water emulsions can contain a combination of surfactantsdescribed herein. For example, a combination of Tween 80 (polysorbate80; polyoxyethuylenesorbitan monooleate) and SPAN85 (sorbitan trioleate)may be used. The emulsions may contain various amounts of Tween 80 andSPAN85 (e.g., those exemplified above) or equal amounts. For example,the oil-in-water emulsions can contain (w/v) about 0.05% Tween 80 andabout 0.05% SPAN85, about 0.1% Tween 80 and about 0.1% SPAN85, about0.2% Tween 80 and about 0.2% SPAN85, about 0.3% Tween 80 and about 0.3%SPAN85, about 0.4% Tween 80 and about 0.4% SPAN85, about 0.5% Tween 80and about 0.5% SPAN85, about 0.6% Tween 80 and about 0.6% SPAN85, about0.7% Tween 80 and about 0.7% SPAN85, about 0.8% Tween 80 and about 0.8%SPAN85, about 0.9% Tween 80 and about 0.9% SPAN85, or about 1% Tween 80and about 1.0% SPAN85.

In certain embodiments, the surfactant is a Polyethylene Glycol(PEG)-lipid. In other embodiments, the emulsion does not comprise aPEG-lipid. PEG-lipids, such as PEG coupled to dialkyloxypropyls(PEG-DAA), PEG coupled to diacylglycerol (PEG-DAG), PEG coupled tophosphatidylethanolamine (PE) (PEG-PE) or some other phospholipids(PEG-phospholipids), PEG conjugated to ceramides (PEG-Cer), or acombination thereof, may also be used as surfactants (see, e.g., U.S.Pat. No. 5,885,613; U.S. patent application publication Nos.2003/0077829, 2005/0175682 and 2006/0025366). Other suitable PEG-lipidsinclude, e.g., PEG-dialkyloxypropyl (DAA) lipids or PEG-diacylglycerol(DAG) lipids. Exemplary PEG-DAG lipids include, e.g.,PEG-dilauroylglycerol (C₁₂) lipids, PEG-dimyristoylglycerol (C₁₄)lipids, PEG-dipalmitoylglycerol (C₁₆) lipids, or PEG-distearoylglycerol(C₁₈) lipids. Exemplary PEG-DAA lipids include, e.g.,PEG-dilauryloxypropyl (C₁₂) lipids, PEG-dimyristyloxypropyl (C₁₄)lipids, PEG-dipalmityloxypropyl (C₁₆) lipids, or PEG-distearyloxypropyl(C₁₈) lipids.

PEGs are classified by their molecular weights; for example, PEG 2000has an average molecular weight of about 2,000 daltons, and PEG 5000 hasan average molecular weight of about 5,000 daltons. PEGs arecommercially available from Sigma Chemical Co. as well as othercompanies and include, for example, the following:monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethyleneglycol-succinate (MePEG-S), monomethoxypolyethylene glycol-succinimidylsuccinate (MePEG-S-NHS), monomethoxypolyethylene glycol-amine(MePEG-NH₂), monomethoxypolyethylene glycol-tresylate (MePEG-TRES), andmonomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM). Inaddition, monomethoxypolyethyleneglycol-acetic acid (MePEG-CH₂COOH), isparticularly useful for preparing the PEG-lipid conjugates including,e.g., PEG-DAA conjugates.

D. Aqueous Phase (Continuous Phase)

The aqueous phase (continuous phase) of the oil-in-water emulsions iswater, or an aqueous solution that can contain a salt (e.g., NaCl), abuffer (e.g., a citrate buffer), a nonionic tonicifying agent (e.g., asaccharide), a polymer, a surfactant, or any combination thereof. Theaqueous phase of the pre-complexed emulsions (oil-in-water emulsionsbefore the addition of the negatively charged molecules) can differ fromthe aqueous phase of the post-complexed emulsions (oil-in-wateremulsions in which the negatively charged molecules are complexed withthe emulsion particles). In general, the pre-complexed emulsions areprepared in an aqueous solvent that promotes the formation of particleswith desired properties (e.g., average diameter, and the like). Thepre-complexed emulsions are diluted with an aqueous solution thatcontains the negatively charged molecule, and other desired components,to produce the final cationic oil-in-water emulsion, which contains thefinal aqueous phase with desired osmolarity and tonicity. The aqueousphase can contain an antioxidant, such as citrate, ascorbate or saltsthereof.

When the emulsions are formulated for in vivo administration, it ispreferable to make up the final solution so that the tonicity andosmolarity of the emulsion are substantially the same as normalphysiological fluids in order to prevent undesired post-administrationconsequences, such as swelling or rapid absorption of the composition.It is also preferable to buffer the aqueous phase in order to maintain apH compatible with normal physiological conditions. Also, in certaininstances, it may be desirable to maintain the pH at a particular levelin order to insure the stability of certain components of the emulsion.For example, it may be desirable to prepare an emulsion that is isotonicand isosmotic. To control tonicity, the emulsion may comprise aphysiological salt, such as a sodium salt. Sodium chloride (NaCl), forexample, may be used at about 0.9% (w/v) (physiological saline). Othersalts that may be present include potassium chloride, potassiumdihydrogen phosphate, disodium phosphate, magnesium chloride, calciumchloride, etc. Non-ionic tonicifying agents can also be used to controltonicity. A number of non-ionic tonicity modifying agents ordinarilyknown to those in the art. These are typically carbohydrates of variousclassifications (see, for example, Voet and Voet (1990) Biochemistry(John Wiley & Sons, New York). Monosaccharides classified as aldosessuch as glucose, mannose, arabinose, and ribose, as well as thoseclassified as ketoses such as fructose, sorbose, and xylulose can beused as non-ionic tonicifying agents in the present invention.Disaccharides such a sucrose, maltose, trehalose, and lactose can alsobe used. In addition, alditols (acyclic polyhydroxy alcohols, alsoreferred to as sugar alcohols) such as glycerol, mannitol, xylitol, andsorbitol are non-ionic tonicifying agents useful in the presentinvention. Non-ionic tonicity modifying agents can be present at aconcentration of from about 0.1% to about 10% or about 1% to about 10%,depending upon the agent that is used.

The aqueous phase may be buffered. Any physiologically acceptable buffermay be used herein, such as water, citrate buffers, phosphate buffers,acetate buffers, tris buffers, bicarbonate buffers, carbonate buffers,succinate buffer, or the like. The pH of the aqueous component willpreferably be between 6.0-8.0, more preferable about 6.2 to about 6.8.In an exemplary embodiment, the buffer is 10 mM citrate buffer with a pHat 6.5. In another exemplary embodiment, the aqueous phase is, or thebuffer prepared using, RNase-free water or DEPC treated water. In somecases, high salt in the buffer might interfere with complexation ofnegatively charged molecule to the emulsion particle therefore isavoided. In other cases, certain amount of salt in the buffer may beincluded.

In an exemplary embodiment, the buffer is 10 mM citrate buffer with a pHat 6.5. If desired the aqueous phase is, or the buffer is preparedusing, RNase-free water or DEPC treated water.

The aqueous phase may also comprise additional components such asmolecules that change the osmolarity of the aqueous phase or moleculesthat stabilizes the negatively charged molecule after complexation.Preferably, the osmolarity of the aqueous phase is adjusted using anon-ionic tonicifying agent, such as a sugar (e.g., trehalose, sucrose,dextrose, fructose, reduced palatinose, etc.), a sugar alcohol (such asmannitol, sorbitol, xylitol, erythritol, lactitol, maltitol, glycerol,etc.). If desired a nonionic polymer polymer (e.g., a poly(alkyl glycol)such as polyethylene glycol, polypropylene glycol, or polybutlyeneglycol) or nonionic surfactant can be used.

In certain embodiments, the aqueous phase of the cationic oil-in-wateremulsion may comprise a polymer or a surfactant, or a combinationthereof. In an exemplary embodiment, the oil-in-water emulsion containsa poloxamer Poloxamers are nonionic triblock copolymers having a centralhydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked bytwo hydrophilic chains of polyoxyethylene (poly(ethylene oxide)).Poloxamers are also known by the trade name Pluronic® polymers.Poloxamer polymers may lead to greater stability and increased RNaseresistance of the RNA molecule after RNA complexation.

Alternatively or in addition, the cationic oil-in-water emulsion maycomprise from about 0.1% to about 20% (w/v) polymer, or from about 0.05%to about 10% (w/v) polymer. For example, the cationic oil-in-wateremulsion may comprise a polymer (e.g., a poloxamer such as Pluronic®F127 ((Ethylene Oxide/Propylene Oxide Block Copolymer:H(OCH₂CH₂)_(x)(OCH₃CH(CH₃))_(y)(OCH₂CH₂)_(z)OH)) at from about 0.1% toabout 20% (w/v), from about 0.1% to about 10% (w/v), from about 0.05% toabout 10% (w/v), or from about 0.05% to about 5% (w/v).

In an exemplary embodiment, the oil-in-water emulsion comprises about 4%(w/v), or about 8% (w/v) Pluronic® F127.

The quantity of the aqueous component employed in these compositionswill be that amount necessary to bring the value of the composition tounity. That is, a quantity of aqueous component sufficient to make 100%will be mixed, with the other components listed above in order to bringthe compositions to volume.

4. Negatively Charged Molecules

When a negatively charged molecule is to be delivered, it can becomplexed with the particles of the cationic oil-in-water emulsions. Thenegatively charged molecule is complexed with the emulsion particles by,for example, interactions between the negatively charged molecule andthe cationic lipid on the surface of the particles, as well ashydrophobic/hydrophilic interactions between the negatively chargedmolecule and the surface of the particles. Although not wishing to bebound by any particular theory, it is believed that the negativelycharged molecules interact with the cationic lipid through non-covalent,ionic charge interactions (electrostatic forces), and the strength ofthe complex as well as the amount of negatively charged compound thatcan be complexed to a particle are related to the amount of cationiclipid in the particle. Additionally, hydrophobic/hydrophilicinteractions between the negatively charged molecule and the surface ofthe particles may also play a role.

Examples of negatively charged molecules include negatively chargedpeptides, polypeptides or proteins, nucleic acid molecules (e.g., singleor double stranded RNA or DNA), small molecules (e.g., small moleculeimmune potentiators (SMIPs), phosphonate, fluorophosphonate, etc.) andthe like. In preferred aspects, the negatively charged molecule is anRNA molecule, such as an RNA that encodes a peptide, polypeptide orprotein, including self-replicating RNA molecules, or a smallinterfering RNA.

The complex can be formed by using techniques known in the art, examplesof which are described herein. For example, a nucleic acid-particlecomplex can be formed by mixing a cationic emulsion with the nucleicacid molecule, for example by vortexing. The amount of the negativelycharged molecule and cationic lipid in the emulsions may be adjusted oroptimized to provide desired strength of binding and binding capacity.For example, as described and exampled herein, exemplary RNA-particlecomplexes were produced by varying the RNA: cationic lipid ratios (asmeasured by the “N/P ratio”). The term N/P ratio refers to the amount(moles) of protonatable nitrogen atoms in the cationic lipid divided bythe amount (moles) of phosphates on the RNA.

Preferred N/P ratios are from about 1:1 to about 20:1, from about 2:1 toabout 18:1, from about 3:1 to 16:1, from about 4:1 to about 14:1, fromabout 6:1 to about 12:1, about 3:1, about 4:1, about 5:1, about 6:1,about 7:1, about 8:1, about 9:1, about 10:1, about 11:1, about 12:1,about 13:1, about 14:1, about 15:1, or about 16:1. Alternatively,preferred N/P ratios are at least about 3:1, at least about 4:1, atleast about 5:1, at least about 6:1, at least about 7:1, at least about8:1, at least about 9:1, at least about 10:1, at least about 11:1, atleast about 12:1, at least about 13:1, at least about 14:1, at leastabout 15:1, or at least about 16:1. A more preferred N/P ratio is about4:1 or higher.

Each emulsion may have its own optimal or preferred N/P ratio to producedesired effects (e.g., desired level of expression of the complexedRNA), which can be determined experimentally (e.g., using the assays asdescribed herein or other techniques known in the art, such as measuringexpression level of a protein that is encoded by the RNA, or measuringthe percentage of the RNA molecules being released from the complex inthe presence of heparin). Generally, the N/P ratio should be at a valuethat at least about 5%, about 10%, about 15%, about 20%, about 25%,about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,or about 95% of the RNA molecules are released from the RNA-particlecomplexes when the RNA-particle complexes are taken up by cells. In someembodiments, the N/P ratio is a value that provides for release of atleast 0.5% or at least 1% of the RNA molecules are released from theRNA-particle complexes when the RNA-particle complexes are taken up bycells.

The expression level of an antigen encoded by the RNA molecule may notnecessarily correlate with the immunogenicity of the antigen. In suchcases, optimal or preferred N/P ratio fore immunogenicity may bedetermined by, e.g., measuring specific antibody titers.

The cationic oil-in-water emulsions described herein are particularlysuitable for formulating nucleic acid-based vaccines (e.g., DNAvaccines, RNA vaccines). The formation of a nucleic acid-emulsionparticle complex facilitates the uptake of the nucleic acid into hostcells, and protects the nucleic acid molecule from nuclease degradation.Transfected cells can then express the antigen encoded by the nucleicacid molecule, which can produce an immune response to the antigen. Likelive or attenuated viruses, nucleic acid-based vaccines can effectivelyengage both MHC-I and MHC-II pathways allowing for the induction of CD8⁺and CD4⁺ T cell responses, whereas antigen present in soluble form, suchas recombinant protein, generally induces only antibody responses.

In certain embodiments, the negatively charged molecule described hereinis an RNA molecule. In certain embodiments, the RNA molecule encodes anantigen (peptide, polypeptide or protein) and the cationic oil in wateremulsion is suitable for use as an RNA-based vaccine. The compositioncan contain more than one species of RNA molecule encoding an antigen,e.g., two, three, five, or ten different species of RNA molecules thatare complexed to the emulsion particles. That is, the composition cancontain one or more different species of RNA molecules, each encoding adifferent antigen. Alternatively or in addition, one RNA molecule mayalso encode more than one antigen, e.g., a bicistronic, or tricistronicRNA molecule that encodes different or identical antigens. Accordingly,the cationic oil in water emulsion is suitable for use as an RNA-basedvaccine, that is monovalent or multivalent. If desired, the RNA moleculecan be polycistronic.

The sequence of the RNA molecule may be codon optimized or deoptimizedfor expression in a desired host, such as a human cell.

The sequence of the RNA molecule may be modified if desired, for exampleto increase the efficacy of expression or replication of the RNA, or toprovide additional stability or resistance to degradation. For example,the RNA sequence can be modified with respect to its codon usage, forexample, to increase translation efficacy and half-life of the RNA. Apoly A tail (e.g., of about 30 adenosine residues or more) (SEQ ID NO:28) may be attached to the 3′ end of the RNA to increase its half-life.The 5′ end of the RNA may be capped with a modified ribonucleotide withthe structure m7G (5′) ppp (5′) N (cap 0 structure) or a derivativethereof, which can be incorporated during RNA synthesis or can beenzymatically engineered after RNA transcription (e.g., by usingVaccinia Virus Capping Enzyme (VCE) consisting of mRNA triphosphatase,guanylyl-transferase and guanine-7-methytransferase, which catalyzes theconstruction of N7-monomethylated cap 0 structures). Cap 0 structureplays an important role in maintaining the stability and translationalefficacy of the RNA molecule. The 5′ cap of the RNA molecule may befurther modified by a 2′-O-Methyltransferase which results in thegeneration of a cap 1 structure (m7Gppp [m2′-O] N), which may furtherincreases translation efficacy.

If desired, the RNA molecule can comprise one or more modifiednucleotides in addition to any 5′ cap structure. There are more than 96naturally occurring nucleoside modifications found on mammalian RNA.See, e.g., Limbach et al., Nucleic Acids Research, 22(12):2183-2196(1994). The preparation of nucleotides and modified nucleotides andnucleosides are well-known in the art, e.g. from U.S. Pat. Nos.4,373,071, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524,5,132,418, 5,153,319, 5,262,530, 5,700,642 all of which are incorporatedby reference in their entirety herein, and many modified nucleosides andmodified nucleotides are commercially available.

Modified nucleobases which can be incorporated into modified nucleosidesand nucleotides and be present in the RNA molecules include: m5C(5-methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), s2U(2-thiouridine), Um (2′-O-methyluridine), m1A (1-methyladenosine); m2A(2-methyladenosine); Am (2-1-O-methyladenosine); ms2m6A(2-methylthio-N6-methyladenosine); i6A (N6-isopentenyladenosine); ms2i6A(2-methylthio-N6isopentenyladenosine); io6A(N6-(cis-hydroxyisopentenyl)adenosine); ms2i6A(2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine); g6A(N6-glycinylcarbamoyladenosine); t6A (N6-threonyl carbamoyladenosine);ms2t6A (2-methylthio-N6-threonyl carbamoyladenosine); m6t6A(N6-methyl-N6-threonylcarbamoyladenosine);hn6A(N6-hydroxynorvalylcarbamoyl adenosine); ms2hn6A(2-methylthio-N6-hydroxynorvalyl carbamoyladenosine); Ar(p)(2′-O-ribosyladenosine (phosphate)); I (inosine); m1I (1-methylinosine);m′Im (1,2′-O-dimethylinosine); m3C (3-methylcytidine); Cm(2T-O-methylcytidine); s2C (2-thiocytidine); ac4C (N4-acetylcytidine);f5C (5-fonnylcytidine); m5Cm (5,2-O-dimethyl cytidine); ac4Cm(N4acetyl2TOmethylcytidine); k2C (lysidine); m1G (1-methylguanosine);m2G (N2-methylguanosine); m7G (7-methylguanosine); Gm(2′-O-methylguanosine); m22G (N2,N2-dimethylguanosine); m2Gm(N2,2′-O-dimethylguanosine); m22Gm (N2,N2,2′-O-trimethylguanosine);Gr(p) (2′-O-ribosylguanosine (phosphate)); yW (wybutosine); o2yW(peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodifiedhydroxywybutosine); imG (wyosine); mimG (methylguanosine); Q(queuosine); oQ (epoxyqueuosine); galQ (galtactosyl-queuosine); manQ(mannosyl-queuosine); preQo (7-cyano-7-deazaguanosine); preQi(7-aminomethyl-7-deazaguanosine); G* (archaeosine); D (dihydrouridine);m5Um (5,2′-O-dimethyluridine); s4U (4-thiouridine); m5s2U(5-methyl-2-thiouridine); s2Um (2-thio-2′-O-methyluridine); acp3U(3-(3-amino-3-carboxypropyl)uridine); hoSU (5-hydroxyuridine); moSU(5-methoxyuridine); cmo5U (uridine 5-oxyacetic acid); mcmo5U (uridine5-oxyacetic acid methyl ester); chm5U(5-(carboxyhydroxymethyl)uridine)); mchm5U(5-(carboxyhydroxymethyl)uridine methyl ester); mcm5U (5-methoxycarbonylmethyluridine); mcm5Um (S-methoxycarbonylmethyl-2-O-methyluridine);mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine); nm5s2U(5-aminomethyl-2-thiouridine); mnm5U (5-methylaminomethyluridine);mnm5s2U (5-methylaminomethyl-2-thiouridine); mnm5se2U(5-methylaminomethyl-2-selenouridine); ncm5U (5-carbamoylmethyluridine); ncm5Um (5-carbamoylmethyl-2′-O-methyluridine); cmnm5U(5-carboxymethylaminomethyluridine); cnmm5Um(5-carboxymethylaminomethyl-2-L-Omethyluridine); cmnm5s2U(5-carboxymethylaminomethyl-2-thiouridine); m62A(N6,N6-dimethyladenosine); Tm (2′-O-methylinosine); m4C(N4-methylcytidine); m4Cm (N4,2-O-dimethylcytidine); hm5C(5-hydroxymethylcytidine); m3U (3-methyluridine); cm5U(5-carboxymethyluridine); m6Am (N6,T-O-dimethyladenosine); rn62Am(N6,N6,O-2-trimethyladenosine); m2′7G (N2,7-dimethylguanosine); m2′2′7G(N2,N2,7-trimethylguanosine); m3Um (3,2T-O-dimethyluridine); m5D(5-methyldihydrouridine); f5Cm (5-formyl-2′-O-methylcytidine); m1Gm(1,2′-O-dimethylguanosine); m′Am (1,2-O-dimethyl adenosine)irinomethyluridine); tm5s2U (S-taurinomethyl-2-thiouridine)); imG-14(4-demethyl guanosine); imG2 (isoguanosine); ac6A (N6-acetyladenosine),hypoxanthine, inosine, 8-oxo-adenine, 7-substituted derivatives thereof,dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil,5-methyluracil, 5-(C₂-C₆)-alkenyluracil, 5-(C₂-C₆)-alkynyluracil,5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil,5-hydroxycytosine, 5-(C₁-C₆)-alkylcytosine, 5-methylcytosine,5-(C₂-C₆)-alkenylcytosine, 5-(C₂-C₆)-alkynylcytosine, 5-chlorocytosine,5-fluorocytosine, 5-bromocytosine, N²-dimethylguanine, 7-deazaguanine,8-azaguanine, 7-deaza-7-substituted guanine,7-deaza-7-(C2-C6)alkynylguanine, 7-deaza-8-substituted guanine,8-hydroxyguanine, 6-thioguanine, 8-oxoguanine, 2-aminopurine,2-amino-6-chloropurine, 2,4-diaminopurine, 2,6-diaminopurine,8-azapurine, substituted 7-deazapurine, 7-deaza-7-substituted purine,7-deaza-8-substituted purine, hydrogen (abasic residue), m5C, m5U, m6A,s2U, W, or 2′-O-methyl-U. Many of these modified nucleobases and theircorresponding ribonucleosides are available from commercial suppliers.See, e.g., WO 2011/005799 which is incorporated herein by reference.

If desired, the RNA molecule can contain phosphoramidate,phosphorothioate, and/or methylphosphonate linkages.

In some embodiments, the RNA molecule does not include modifiednucleotides, e.g., does not include modified nucleobases, and all of thenucleotides in the RNA molecule are conventional standardribonucleotides A, U, G and C, with the exception of an optional 5′ capthat may include, for example, 7-methylguanosine. In other embodiments,the RNA may include a 5′ cap comprising a 7′-methylguanosine, and thefirst 1, 2 or 3 5′ ribonucleotides may be methylated at the 2′ positionof the ribose.

A. Self-Replicating RNA

In some aspects, the cationic oil in water emulsion contains aself-replicating RNA molecule. In certain embodiments, theself-replicating RNA molecule is derived from or based on an alphavirus.

Self-replicating RNA molecules are well known in the art and can beproduced by using replication elements derived from, e.g., alphaviruses,and substituting the structural viral proteins with a nucleotidesequence encoding a protein of interest. Cells transfected withself-replicating RNA briefly produce antigen before undergoing apoptoticdeath. This death is a likely result of requisite double-stranded (ds)RNA intermediates, which also have been shown to super-activateDendritic Cells. Thus, the enhanced immunogenicity of self-replicatingRNA may be a result of the production of pro-inflammatory dsRNA, whichmimics an RNA-virus infection of host cells.

Advantageously, the cell's machinery is used by self-replicating RNAmolecules to generate an exponential increase of encoded gene products,such as proteins or antigens, which can accumulate in the cells or besecreted from the cells. Overexpression of proteins or antigens byself-replicating RNA molecules takes advantage of the immunostimulatoryadjuvant effects, including stimulation of toll-like receptors (TLR) 3,7 and 8 and non TLR pathways (e.g, RIG-1, MD-5) by the products of RNAreplication and amplification, and translation which induces apoptosisof the transfected cell.

The self-replicating RNA generally contains at least one or more genesselected from the group consisting of viral replicases, viral proteases,viral helicases and other nonstructural viral proteins, and alsocomprise 5′- and 3′-end cis-active replication sequences, and ifdesired, a heterologous sequences that encode a desired amino acidsequences (e.g., an antigen of interest). A subgenomic promoter thatdirects expression of the heterologous sequence can be included in theself-replicating RNA. If desired, the heterologous sequence (e.g., anantigen of interest) may be fused in frame to other coding regions inthe self-replicating RNA and/or may be under the control of an internalribosome entry site (IRES).

In certain embodiments, the self-replicating RNA molecule is notencapsulated in a virus-like particle. Self-replicating RNA molecules ofthe invention can be designed so that the self-replicating RNA moleculecannot induce production of infectious viral particles. This can beachieved, for example, by omitting one or more viral genes encodingstructural proteins that are necessary for the production of viralparticles in the self-replicating RNA. For example, when theself-replicating RNA molecule is based on an alpha virus, such asSinebis virus (SIN), Semliki forest virus and Venezuelan equineencephalitis virus (VEE), one or more genes encoding viral structuralproteins, such as capsid and/or envelope glycoproteins, can be omitted.

If desired, self-replicating RNA molecules of the invention can also bedesigned to induce production of infectious viral particles that areattenuated or virulent, or to produce viral particles that are capableof a single round of subsequent infection.

When delivered to a vertebrate cell, a self-replicating RNA molecule canlead to the production of multiple daughter RNAs by transcription fromitself (or from an antisense copy of itself). The self-replicating RNAcan be directly translated after delivery to a cell, and thistranslation provides a RNA-dependent RNA polymerase which then producestranscripts from the delivered RNA. Thus the delivered RNA leads to theproduction of multiple daughter RNAs. These transcripts are antisenserelative to the delivered RNA and may be translated themselves toprovide in situ expression of a gene product, or may be transcribed toprovide further transcripts with the same sense as the delivered RNAwhich are translated to provide in situ expression of the gene product.

One suitable system for achieving self-replication is to use analphavirus-based RNA replicon. Alphaviruses comprise a set ofgenetically, structurally, and serologically related arthropod-borneviruses of the Togaviridae family Twenty-six known viruses and virussubtypes have been classified within the alphavirus genus, including,Sindbis virus, Semliki Forest virus, Ross River virus, and Venezuelanequine encephalitis virus. As such, the self-replicating RNA of theinvention may incorporate a RNA replicase derived from semliki forestvirus (SFV), sindbis virus (SIN), Venezuelan equine encephalitis virus(VEE), Ross-River virus (RRV), or other viruses belonging to thealphavirus family.

An alphavirus-based “replicon” expression vectors can be used in theinvention. Replicon vectors may be utilized in several formats,including DNA, RNA, and recombinant replicon particles. Such repliconvectors have been derived from alphaviruses that include, for example,Sindbis virus (Xiong et al. (1989) Science 243:1188-1191; Dubensky etal., (1996) J. Virol. 70:508-519; Hariharan et al. (1998) J. Virol.72:950-958; Polo et al. (1999) PNAS 96:4598-4603), Semliki Forest virus(Liljestrom (1991) Bio/Technology 9:1356-1361; Berglund et al. (1998)Nat. Biotech. 16:562-565), and Venezuelan equine encephalitis virus(Pushko et al. (1997) Virology 239:389-401). Alphaviruses-derivedreplicons are generally quite similar in overall characteristics (e.g.,structure, replication), individual alphaviruses may exhibit someparticular property (e.g., receptor binding, interferon sensitivity, anddisease profile) that is unique. Therefore, chimeric alphavirusreplicons made from divergent virus families may also be useful.

Alphavirus-based replicons are (+)-stranded replicons that can betranslated after delivery to a cell to give of a replicase (orreplicase-transcriptase). The replicase is translated as a polyproteinwhich auto-cleaves to provide a replication complex which createsgenomic (−)-strand copies of the +-strand delivered RNA. These(−)-strand transcripts can themselves be transcribed to give furthercopies of the (+)-stranded parent RNA and also to give a subgenomictranscript which encodes the desired gene product. Translation of thesubgenomic transcript thus leads to in situ expression of the desiredgene product by the infected cell. Suitable alphavirus replicons can usea replicase from a sindbis virus, a semliki forest virus, an easternequine encephalitis virus, a venezuelan equine encephalitis virus, etc.

A preferred self-replicating RNA molecule thus encodes (i) aRNA-dependent RNA polymerase which can transcribe RNA from theself-replicating RNA molecule and (ii) a polypeptide antigen. Thepolymerase can be an alphavirus replicase e.g. comprising alphavirusprotein nsP4.

Whereas natural alphavirus genomes encode structural virion proteins inaddition to the non-structural replicase, it is preferred that analphavirus based self-replicating RNA molecule of the invention does notencode alphavirus structural proteins. Thus the self-replicating RNA canlead to the production of genomic RNA copies of itself in a cell, butnot to the production of RNA-containing alphavirus virions. Theinability to produce these virions means that, unlike a wild-typealphavirus, the self-replicating RNA molecule cannot perpetuate itselfin infectious form. The alphavirus structural proteins which arenecessary for perpetuation in wild-type viruses are absent fromself-replicating RNAs of the invention and their place is taken bygene(s) encoding the desired gene product, such that the subgenomictranscript encodes the desired gene product rather than the structuralalphavirus virion proteins.

Thus a self-replicating RNA molecule useful with the invention may havetwo open reading frames. The first (5′) open reading frame encodes areplicase; the second (3′) open reading frame encodes a polypeptideantigen. In some embodiments the RNA may have additional (downstream)open reading frames e.g. that encode another desired gene products. Aself-replicating RNA molecule can have a 5′ sequence which is compatiblewith the encoded replicase.

In other aspects, the self-replicating RNA molecule is derived from orbased on a virus other than an alphavirus, preferably, apositive-stranded RNA virus, and more preferably a picornavirus,flavivirus, rubivirus, pestivirus, hepacivirus, calicivirus, orcoronavirus. Suitable wild-type alphavirus sequences are well-known andare available from sequence depositories, such as the American TypeCulture Collection, Rockville, Md. Representative examples of suitablealphaviruses include Aura (ATCC VR-368), Bebaru virus (ATCC VR-600, ATCCVR-1240), Cabassou (ATCC VR-922), Chikungunya virus (ATCC VR-64, ATCCVR-1241), Eastern equine encephalomyelitis virus (ATCC VR-65, ATCCVR-1242), Fort Morgan (ATCC VR-924), Getah virus (ATCC VR-369, ATCCVR-1243), Kyzylagach (ATCC VR-927), Mayaro (ATCC VR-66), Mayaro virus(ATCC VR-1277), Middleburg (ATCC VR-370), Mucambo virus (ATCC VR-580,ATCC VR-1244), Ndumu (ATCC VR-371), Pixuna virus (ATCC VR-372, ATCCVR-1245), Ross River virus (ATCC VR-373, ATCC VR-1246), Semliki Forest(ATCC VR-67, ATCC VR-1247), Sindbis virus (ATCC VR-68, ATCC VR-1248),Tonate (ATCC VR-925), Triniti (ATCC VR-469), Una (ATCC VR-374),Venezuelan equine encephalomyelitis (ATCC VR-69, ATCC VR-923, ATCCVR-1250 ATCC VR-1249, ATCC VR-532), Western equine encephalomyelitis(ATCC VR-70, ATCC VR-1251, ATCC VR-622, ATCC VR-1252), Whataroa (ATCCVR-926), and Y-62-33 (ATCC VR-375).

The self-replicating RNA molecules of the invention are larger thanother types of RNA (e.g. mRNA). Typically, the self-replicating RNAmolecules of the invention contain at least about 4 kb. For example, theself-replicating RNA can contain at least about 5 kb, at least about 6kb, at least about 7 kb, at least about 8 kb, at least about 9 kb, atleast about 10 kb, at least about 11 kb, at least about 12 kb or morethan 12 kb. In certain examples, the self-replicating RNA is about 4 kbto about 12 kb, about 5 kb to about 12 kb, about 6 kb to about 12 kb,about 7 kb to about 12 kb, about 8 kb to about 12 kb, about 9 kb toabout 12 kb, about 10 kb to about 12 kb, about 11 kb to about 12 kb,about 5 kb to about 11 kb, about 5 kb to about 10 kb, about 5 kb toabout 9 kb, about 5 kb to about 8 kb, about 5 kb to about 7 kb, about 5kb to about 6 kb, about 6 kb to about 12 kb, about 6 kb to about 11 kb,about 6 kb to about 10 kb, about 6 kb to about 9 kb, about 6 kb to about8 kb, about 6 kb to about 7 kb, about 7 kb to about 11 kb, about 7 kb toabout 10 kb, about 7 kb to about 9 kb, about 7 kb to about 8 kb, about 8kb to about 11 kb, about 8 kb to about 10 kb, about 8 kb to about 9 kb,about 9 kb to about 11 kb, about 9 kb to about 10 kb, or about 10 kb toabout 11 kb.

The self-replicating RNA molecules of the invention may comprise one ormore modified nucleotides (e.g., pseudouridine, N6-methyladenosine,5-methylcytidine, 5-methyluridine).

The self-replicating RNA molecule may encode a single polypeptideantigen or, optionally, two or more of polypeptide antigens linkedtogether in a way that each of the sequences retains its identity (e.g.,linked in series) when expressed as an amino acid sequence. Thepolypeptides generated from the self-replicating RNA may then beproduced as a fusion polypeptide or engineered in such a manner toresult in separate polypeptide or peptide sequences.

The self-replicating RNA of the invention may encode one or morepolypeptide antigens that contain a range of epitopes. Preferablyepitopes capable of eliciting either a helper T-cell response or acytotoxic T-cell response or both.

The self-replicating RNA molecules described herein may be engineered toexpress multiple nucleotide sequences, from two or more open readingframes, thereby allowing co-expression of proteins, such as a two ormore antigens together with cytokines or other immunomodulators, whichcan enhance the generation of an immune response. Such aself-replicating RNA molecule might be particularly useful, for example,in the production of various gene products (e.g., proteins) at the sametime, for example, as a bivalent or multivalent vaccine.

The self-replicating RNA molecules of the invention can be preparedusing any suitable method. Several suitable methods are known in the artfor producing RNA molecules that contain modified nucleotides. Forexample, a self-replicating RNA molecule that contains modifiednucleotides can be prepared by transcribing (e.g., in vitrotranscription) a DNA that encodes the self-replicating RNA moleculeusing a suitable DNA-dependent RNA polymerase, such as T7 phage RNApolymerase, SP6 phage RNA polymerase, T3 phage RNA polymerase, and thelike, or mutants of these polymerases which allow efficientincorporation of modified nucleotides into RNA molecules. Thetranscription reaction will contain nucleotides and modifiednucleotides, and other components that support the activity of theselected polymerase, such as a suitable buffer, and suitable salts. Theincorporation of nucleotide analogs into a self-replicating RNA may beengineered, for example, to alter the stability of such RNA molecules,to increase resistance against RNases, to establish replication afterintroduction into appropriate host cells (“infectivity” of the RNA),and/or to induce or reduce innate and adaptive immune responses.

Suitable synthetic methods can be used alone, or in combination with oneor more other methods (e.g., recombinant DNA or RNA technology), toproduce a self-replicating RNA molecule of the invention. Suitablemethods for de novo synthesis are well-known in the art and can beadapted for particular applications. Exemplary methods include, forexample, chemical synthesis using suitable protecting groups such as CEM(Masuda et al., (2007) Nucleic Acids Symposium Series 51:3-4), theβ-cyanoethyl phosphoramidite method (Beaucage S L et al. (1981)Tetrahedron Lett 22:1859); nucleoside H-phosphonate method (GareggPetal. (1986) Tetrahedron Lett 27:4051-4; Froehler B C et al. (1986)Nucl Acid Res 14:5399-407; Garegg Petal. (1986) Tetrahedron Lett27:4055-8; Gaffney B L et al. (1988) Tetrahedron Lett 29:2619-22). Thesechemistries can be performed or adapted for use with automated nucleicacid synthesizers that are commercially available. Additional suitablesynthetic methods are disclosed in Uhlmann et al. (1990) Chem Rev90:544-84, and Goodchild J (1990) Bioconjugate Chem 1: 165. Nucleic acidsynthesis can also be performed using suitable recombinant methods thatare well-known and conventional in the art, including cloning,processing, and/or expression of polynucleotides and gene productsencoded by such polynucleotides. DNA shuffling by random fragmentationand PCR reassembly of gene fragments and synthetic polynucleotides areexamples of known techniques that can be used to design and engineerpolynucleotide sequences. Site-directed mutagenesis can be used to alternucleic acids and the encoded proteins, for example, to insert newrestriction sites, alter glycosylation patterns, change codonpreference, produce splice variants, introduce mutations and the like.Suitable methods for transcription, translation and expression ofnucleic acid sequences are known and conventional in the art. (Seegenerally, Current Protocols in Molecular Biology, Vol. 2, Ed. Ausubel,et al., Greene Publish. Assoc. & Wiley Interscience, Ch. 13, 1988;Glover, DNA Cloning, Vol. II, IRL Press, Wash., D.C., Ch. 3, 1986;Bitter, et al., in Methods in Enzymology 153:516-544 (1987); TheMolecular Biology of the Yeast Saccharomyces, Eds. Strathern et al.,Cold Spring Harbor Press, Vols. I and II, 1982; and Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989.)

The presence and/or quantity of one or more modified nucleotides in aself-replicating RNA molecule can be determined using any suitablemethod. For example, a self-replicating RNA can be digested tomonophosphates (e.g., using nuclease P1) and dephosphorylated (e.g.,using a suitable phosphatase such as CIAP), and the resultingnucleosides analyzed by reversed phase HPLC (e.g., usings a YMC PackODS-AQ column (5 micron, 4.6×250 mm) and elute using a gradient, 30% B(0-5 min) to 100% B (5-13 min) and at 100% B (13-40) min, flow Rate (0.7ml/min), UV detection (wavelength: 260 nm), column temperature (30° C.).Buffer A (20 mM acetic acid-ammonium acetate pH 3.5), buffer B (20 mMacetic acid-ammonium acetate pH 3.5/methanol [90/10])).

Optionally, the self-replicating RNA molecules of the invention mayinclude one or more modified nucleotides so that the self-replicatingRNA molecule will have less immunomodulatory activity upon introductionor entry into a host cell (e.g., a human cell) in comparison to thecorresponding self-replicating RNA molecule that does not containmodified nucleotides.

If desired, the self-replicating RNA molecules can be screened oranalyzed to confirm their therapeutic and prophylactic properties usingvarious in vitro or in vivo testing methods that are known to those ofskill in the art. For example, vaccines comprising self-replicating RNAmolecule can be tested for their effect on induction of proliferation oreffector function of the particular lymphocyte type of interest, e.g., Bcells, T cells, T cell lines, and T cell clones. For example, spleencells from immunized mice can be isolated and the capacity of cytotoxicT lymphocytes to lyse autologous target cells that contain a selfreplicating RNA molecule that encodes a polypeptide antigen. Inaddition, T helper cell differentiation can be analyzed by measuringproliferation or production of TH1 (IL-2 and IFN-γ) and/or TH2 (IL-4 andIL-5) cytokines by ELISA or directly in CD4+ T cells by cytoplasmiccytokine staining and flow cytometry.

Self-replicating RNA molecules that encode a polypeptide antigen canalso be tested for ability to induce humoral immune responses, asevidenced, for example, by induction of B cell production of antibodiesspecific for an antigen of interest. These assays can be conductedusing, for example, peripheral B lymphocytes from immunized individuals.Such assay methods are known to those of skill in the art. Other assaysthat can be used to characterize the self-replicating RNA molecules ofthe invention can involve detecting expression of the encoded antigen bythe target cells. For example, FACS can be used to detect antigenexpression on the cell surface or intracellularly. Another advantage ofFACS selection is that one can sort for different levels of expression;sometimes-lower expression may be desired. Other suitable method foridentifying cells which express a particular antigen involve panningusing monoclonal antibodies on a plate or capture using magnetic beadscoated with monoclonal antibodies.

B. Antigens

In certain embodiments, the negatively charged molecule described hereinis a nucleic acid molecule (e.g., an RNA molecule) that encodes anantigen. Suitable antigens include, but are not limited to, a bacterialantigen, a viral antigen, a fungal antigen, a protazoan antigen, a plantantigen, a cancer antigen, or a combination thereof.

Suitable antigens include proteins and peptides from a pathogen such asa virus, bacteria, fungus, protozoan, plant or from a tumor. Viralantigens and immunogens that can be encoded by the self-replicating RNAmolecule include, but are not limited to, proteins and peptides from aOrthomyxoviruses, such as Influenza A, B and C; Paramyxoviridae viruses,such as Pneumoviruses (RSV), Paramyxoviruses (PIV), Metapneumovirus andMorbilliviruses (e.g., measles); Pneumoviruses, such as Respiratorysyncytial virus (RSV), Bovine respiratory syncytial virus, Pneumoniavirus of mice, and Turkey rhinotracheitis virus; Paramyxoviruses, suchas Parainfluenza virus types 1-4 (PIV), Mumps virus, Sendai viruses,Simian virus 5, Bovine parainfluenza virus, Nipahvirus, Henipavirus andNewcastle disease virus; Poxviridae, including a Orthopoxvirus such asVariola vera (including but not limited to, Variola major and Variolaminor); Metapneumoviruses, such as human metapneumovirus (hMPV) andavian metapneumoviruses (aMPV); Morbilliviruses, such as Measles;Picornaviruses, such as Enteroviruses, Rhinoviruses, Heparnavirus,Parechovirus, Cardioviruses and Aphthoviruses; Enteroviruseses, such asPoliovirus types 1, 2 or 3, Coxsackie A virus types 1 to 22 and 24,Coxsackie B virus types 1 to 6, Echovirus (ECHO) virus types 1 to 9, 11to 27 and 29 to 34 and Enterovirus 68 to 71, Bunyaviruses, including aOrthobunyavirus such as California encephalitis virus; a Phlebovirus,such as Rift Valley Fever virus; a Nairovirus, such as Crimean-Congohemorrhagic fever virus; Heparnaviruses, such as, Hepatitis A virus(HAV); Togaviruses (Rubella), such as a Rubivirus, an Alphavirus, or anArterivirus; Flaviviruses, such as Tick-borne encephalitis (TBE) virus,Dengue (types 1, 2, 3 or 4) virus, Yellow Fever virus, Japaneseencephalitis virus, Kyasanur Forest Virus, West Nile encephalitis virus,St. Louis encephalitis virus, Russian spring-summer encephalitis virus,Powassan encephalitis virus; Pestiviruses, such as Bovine viral diarrhea(BVDV), Classical swine fever (CSFV) or Border disease (BDV);Hepadnaviruses, such as Hepatitis B virus, Hepatitis C virus;Rhabdoviruses, such as a Lyssavirus (Rabies virus) and Vesiculovirus(VSV), Caliciviridae, such as Norwalk virus, and Norwalk-like Viruses,such as Hawaii Virus and Snow Mountain Virus; Coronaviruses, such asSARS, Human respiratory coronavirus, Avian infectious bronchitis (IBV),Mouse hepatitis virus (MHV), and Porcine transmissible gastroenteritisvirus (TGEV); Retroviruses such as an Oncovirus, a Lentivirus or aSpumavirus; Reoviruses, as an Orthoreovirus, a Rotavirus, an Orbivirus,or a Coltivirus; Parvoviruses, such as Parvovirus B19; Delta hepatitisvirus (HDV); Hepatitis E virus (HEV); Hepatitis G virus (HGV); HumanHerpesviruses, such as, by way Herpes Simplex Viruses (HSV),Varicella-zoster virus (VZV), Epstein-Barr virus (EBV), Cytomegalovirus(CMV), Human Herpesvirus 6 (HHV6), Human Herpesvirus 7 (HHV7), and HumanHerpesvirus 8 (HHV8); Papovaviruses, such as Papillomaviruses andPolyomaviruses, Adenoviruess and Arenaviruses.

In some embodiments, the antigen elicits an immune response against avirus which infects fish, such as: infectious salmon anemia virus(ISAV), salmon pancreatic disease virus (SPDV), infectious pancreaticnecrosis virus (IPNV), channel catfish virus (CCV), fish lymphocystisdisease virus (FLDV), infectious hematopoietic necrosis virus (IHNV),koi herpesvirus, salmon picorna-like virus (also known as picorna-likevirus of atlantic salmon), landlocked salmon virus (LSV), atlanticsalmon rotavirus (ASR), trout strawberry disease virus (TSD), cohosalmon tumor virus (CSTV), or viral hemorrhagic septicemia virus (VHSV).

In some embodiments the antigen elicits an immune response against aparasite from the Plasmodium genus, such as P. falciparum, P. vivax, P.malariae or P. ovale. Thus the invention may be used for immunizingagainst malaria. In some embodiments the antigen elicits an immuneresponse against a parasite from the Caligidae family, particularlythose from the Lepeophtheirus and Caligus genera e.g. sea lice such asLepeophtheirus salmonis or Caligus rogercresseyi.

Bacterial antigens and immunogens that can be encoded by theself-replicating RNA molecule include, but are not limited to, proteinsand peptides from Neisseria meningitides, Streptococcus pneumoniae,Streptococcus pyogenes, Moraxella catarrhalis, Bordetella pertussis,Burkholderia sp. (e.g., Burkholderia mallei, Burkholderia pseudomalleiand Burkholderia cepacia), Staphylococcus aureus, Staphylococcusepidermis, Haemophilus influenzae, Clostridium tetani (Tetanus),Clostridium perfringens, Clostridium botulinums (Botulism),Cornynebacterium diphtherias (Diphtheria), Pseudomonas aeruginosa,Legionella pneumophila, Coxiella burnetii, Brucella sp. (e.g., B.abortus, B. canis, B. melitensis, B. neotomae, B. ovis, B. suis and B.pinnipediae), Francisella sp. (e.g., F. novicida, F. philomiragia and F.tularensis), Streptococcus agalactiae, Neiserria gonorrhoeae, Chlamydiatrachomatis, Treponema pallidum (Syphilis), Haemophilus ducreyi,Enterococcus faecalis, Enterococcus faecium, Helicobacter pylori,Staphylococcus saprophyticus, Yersinia enterocolitica, E. coli (such asenterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAggEC),diffusely adhering E. coli (DAEC), enteropathogenic E. coli (EPEC),extraintestinal pathogenic E. coli (ExPEC; such as uropathogenic E. coli(UPEC) and meningitis/sepsis-associated E. coli (MNEC)), and/orenterohemorrhagic E. coli (EHEC), Bacillus anthracia (anthrax), Yersiniapestis (plague), Mycobacterium tuberculosis, Rickettsia, Listeriamonocytogenes, Chlamydia pneumoniae, Vibrio cholerae, Salmonella typhi(typhoid fever), Borrelia burgdorfer, Porphyromonas gingivalis,Klebsiella, Mycoplasma pneumoniae, etc.

Fungal antigens and immunogens that can be encoded by theself-replicating RNA molecule include, but are not limited to, proteinsand peptides from Dermatophytres, including: Epidermophyton floccusum,Microsporum audouini, Microsporum canis, Microsporum distortum,Microsporum equinum, Microsporum gypsum, Microsporum nanum, Trichophytonconcentricum, Trichophyton equinum, Trichophyton gallinae, Trichophytongypseum, Trichophyton megnini, Trichophyton mentagrophytes, Trichophytonquinckeanum, Trichophyton rubrum, Trichophyton schoenleini, Trichophytontonsurans, Trichophyton verrucosum, T verrucosum var. album, var.discoides, var. ochraceum, Trichophyton violaceum, and/or Trichophytonfaviforme; or from Aspergillus fumigatus, Aspergillus flavus,Aspergillus niger, Aspergillus nidulans, Aspergillus terreus,Aspergillus sydowii, Aspergillus flavatus, Aspergillus glaucus,Blastoschizomyces capitatus, Candida albicans, Candida enolase, Candidatropicalis, Candida glabrata, Candida krusei, Candida parapsilosis,Candida stellatoidea, Candida kusei, Candida parakwsei, Candidalusitaniae, Candida pseudotropicalis, Candida guilliermondi,Cladosporium carrionii, Coccidioides immitis, Blastomyces dermatidis,Cryptococcus neoformans, Geotrichum clavatum, Histoplasma capsulatum,Klebsiella pneumoniae, Microsporidia, Encephalitozoon spp., Septataintestinalis and Enterocytozoon bieneusi; the less common are Brachiolaspp, Microsporidium spp., Nosema spp., Pleistophora spp.,Trachipleistophora spp., Vittaforma spp Paracoccidioides brasiliensis,Pneumocystis carinii, Pythiumn insidiosum, Pityrosporum ovale,Sacharomyces cerevisae, Saccharomyces boulardii, Saccharomyces pombe,Scedosporium apiosperum, Sporothrix schenckii, Trichosporon beigelii,Toxoplasma gondii, Penicillium mameffei, Malassezia spp., Fonsecaeaspp., Wangiella spp., Sporothrix spp., Basidiobolus spp., Conidiobolusspp., Rhizopus spp, Mucor spp, Absidia spp, Mortierella spp,Cunninghamella spp, Saksenaea spp., Alternaria spp, Curvularia spp,Helminthosporium spp, Fusarium spp, Aspergillus spp, Penicillium spp,Monolinia spp, Rhizoctonia spp, Paecilomyces spp, Pithomyces spp, andCladosporium spp.

Protazoan antigens and immunogens that can be encoded by theself-replicating RNA molecule include, but are not limited to, proteinsand peptides from Entamoeba histolytica, Giardia lambli, Cryptosporidiumparvum, Cyclospora cayatanensis and Toxoplasma.

Plant antigens and immunogens that can be encoded by theself-replicating RNA molecule include, but are not limited to, proteinsand peptides from Ricinus communis.

Suitable antigens include proteins and peptides from a virus such as,for example, human immunodeficiency virus (HIV), hepatitis A virus(HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplexvirus (HSV), cytomegalovirus (CMV), influenza virus (flu), respiratorysyncytial virus (RSV), parvovorus, norovirus, human papilloma virus(HPV), rhinovirus, yellow fever virus, rabies virus, Dengue fever virus,measles virus, mumps virus, rubella virus, varicella zoster virus,enterovirus (e.g., enterovirus 71), ebola virus, and bovine diarrheavirus. Preferably, the antigenic substance is selected from the groupconsisting of HSV glycoprotein gD, HIV glycoprotein gp120, HIVglycoprotein gp 40, HIV p55 gag, and polypeptides from the pol and tatregions. In other preferred embodiments of the invention, the antigen isa protein or peptide derived from a bacterium such as, for example,Helicobacter pylori, Haemophilus influenza, Vibrio cholerae (cholera),C. diphtherias (diphtheria), C. tetani (tetanus), Neisseriameningitidis, B. pertussis, Mycobacterium tuberculosis, and the like.

HIV antigens that can be encoded by the self-replicating RNA moleculesof the invention are described in U.S. application Ser. No. 490,858,filed Mar. 9, 1990, and published European application number 181150(May 14, 1986), as well as U.S. application Ser. Nos. 60/168,471;09/475,515; 09/475,504; and Ser. No. 09/610,313, the disclosures ofwhich are incorporated herein by reference in their entirety.

Cytomegalovirus antigens that can be encoded by the self-replicating RNAmolecules of the invention are described in U.S. Pat. No. 4,689,225,U.S. application Ser. No. 367,363, filed Jun. 16, 1989 and PCTPublication WO 89/07143, the disclosures of which are incorporatedherein by reference in their entirety.

Hepatitis C antigens that can be encoded by the self-replicating RNAmolecules of the invention are described in PCT/US88/04125, publishedEuropean application number 318216 (May 31, 1989), published Japaneseapplication number 1-500565 filed Nov. 18, 1988, Canadian application583,561, and EPO 388,232, disclosures of which are incorporated hereinby reference in their entirety. A different set of HCV antigens isdescribed in European patent application 90/302866.0, filed Mar. 16,1990, and U.S. application Ser. No. 456,637, filed Dec. 21, 1989, andPCT/US90/01348, the disclosures of which are incorporated herein byreference in their entirety.

In some embodiments, the antigen is derived from an allergen, such aspollen allergens (tree-, herb, weed-, and grass pollen allergens);insect or arachnid allergens (inhalant, saliva and venom allergens, e.g.mite allergens, cockroach and midges allergens, hymenopthera venomallergens); animal hair and dandruff allergens (from e.g. dog, cat,horse, rat, mouse, etc.); and food allergens (e.g. a gliadin). Importantpollen allergens from trees, grasses and herbs are such originating fromthe taxonomic orders of Fagales, Oleales, Pinales and platanaceaeincluding, but not limited to, birch (Betula), alder (Alnus), hazel(Corylus), hornbeam (Carpinus) and olive (Olea), cedar (Cryptomeria andJuniperus), plane tree (Platanus), the order of Poales including grassesof the genera Lolium, Phleum, Poa, Cynodon, Dactylis, Holcus, Phalaris,Secale, and Sorghum, the orders of Asterales and Urticales includingherbs of the genera Ambrosia, Artemisia, and Parietaria. Other importantinhalation allergens are those from house dust mites of the genusDermatophagoides and Euroglyphus, storage mite e.g. Lepidoglyphys,Glycyphagus and Tyrophagus, those from cockroaches, midges and flease.g. Blatella, Periplaneta, Chironomus and Ctenocepphalides, and thosefrom mammals such as cat, dog and horse, venom allergens including suchoriginating from stinging or biting insects such as those from thetaxonomic order of Hymenoptera including bees (Apidae), wasps(Vespidea), and ants (Formicoidae).

In certain embodiments, a tumor immunogen or antigen, or cancerimmunogen or antigen, can be encoded by the self-replicating RNAmolecule. In certain embodiments, the tumor immunogens and antigens arepeptide-containing tumor antigens, such as a polypeptide tumor antigenor glycoprotein tumor antigens.

Tumor immunogens and antigens appropriate for the use herein encompass awide variety of molecules, such as (a) polypeptide-containing tumorantigens, including polypeptides (which can range, for example, from8-20 amino acids in length, although lengths outside this range are alsocommon), lipopolypeptides and glycoproteins.

In certain embodiments, tumor immunogens are, for example, (a) fulllength molecules associated with cancer cells, (b) homologs and modifiedforms of the same, including molecules with deleted, added and/orsubstituted portions, and (c) fragments of the same. Tumor immunogensinclude, for example, class I-restricted antigens recognized by CD8+lymphocytes or class II-restricted antigens recognized by CD4+lymphocytes.

In certain embodiments, tumor immunogens include, but are not limitedto, (a) cancer-testis antigens such as NY-ESO-1, SSX2, SCP1 as well asRAGE, BAGE, GAGE and MAGE family polypeptides, for example, GAGE-1,GAGE-2, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE-12(which can be used, for example, to address melanoma, lung, head andneck, NSCLC, breast, gastrointestinal, and bladder tumors), (b) mutatedantigens, for example, p53 (associated with various solid tumors, e.g.,colorectal, lung, head and neck cancer), p21/Ras (associated with, e.g.,melanoma, pancreatic cancer and colorectal cancer), CDK4 (associatedwith, e.g., melanoma), MUM1 (associated with, e.g., melanoma), caspase-8(associated with, e.g., head and neck cancer), CIA 0205 (associatedwith, e.g., bladder cancer), HLA-A2-R1701, beta catenin (associatedwith, e.g., melanoma), TCR (associated with, e.g., T-cell non-Hodgkinslymphoma), BCR-abl (associated with, e.g., chronic myelogenousleukemia), triosephosphate isomerase, KIA 0205, CDC-27, and LDLR-FUT,(c) over-expressed antigens, for example, Galectin 4 (associated with,e.g., colorectal cancer), Galectin 9 (associated with, e.g., Hodgkin'sdisease), proteinase 3 (associated with, e.g., chronic myelogenousleukemia), WT 1 (associated with, e.g., various leukemias), carbonicanhydrase (associated with, e.g., renal cancer), aldolase A (associatedwith, e.g., lung cancer), PRAME (associated with, e.g., melanoma),HER-2/neu (associated with, e.g., breast, colon, lung and ovariancancer), alpha-fetoprotein (associated with, e.g., hepatoma), KSA(associated with, e.g., colorectal cancer), gastrin (associated with,e.g., pancreatic and gastric cancer), telomerase catalytic protein,MUC-1 (associated with, e.g., breast and ovarian cancer), G-250(associated with, e.g., renal cell carcinoma), p53 (associated with,e.g., breast, colon cancer), and carcinoembryonic antigen (associatedwith, e.g., breast cancer, lung cancer, and cancers of thegastrointestinal tract such as colorectal cancer), (d) shared antigens,for example, melanoma-melanocyte differentiation antigens such asMART-1/Melan A, gp100, MC1R, melanocyte-stimulating hormone receptor,tyrosinase, tyrosinase related protein-1/TRP1 and tyrosinase relatedprotein-2/TRP2 (associated with, e.g., melanoma), (e) prostateassociated antigens such as PAP, PSA, PSMA, PSH-P1, PSM-P1, PSM-P2,associated with e.g., prostate cancer, (f) immunoglobulin idiotypes(associated with myeloma and B cell lymphomas, for example).

In certain embodiments, tumor immunogens include, but are not limitedto, p15, Hom/Mel-40, H-Ras, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, EpsteinBarr virus antigens, EBNA, human papillomavirus (HPV) antigens,including E6 and E7, hepatitis B and C virus antigens, human T-celllymphotropic virus antigens, TSP-180, p185erbB2, p180erbB-3, c-met,mn-23H1, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, p16, TAGE,PSCA, CT7, 43-9F, 5T4, 791 Tgp72, beta-HCG, BCA225, BTAA, CA 125, CA15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029,FGF-5, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K,NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein\cyclophilinC-associated protein), TAAL6, TAG72, TLP, TPS, and the like.

C. Formulations for the Negatively Charged Molecule

The negatively charged molecule (such as RNA) is generally provided inthe form of an aqueous solution, or a form that can be readily dissolvedin an aqueous solution (e.g., lyophilized). The aqueous solution can bewater, or an aqueous solution that comprises a salt (e.g., NaCl), abuffer (e.g., a citrate buffer), a nonionic tonicifying agent (e.g., asaccharide), a polymer, a surfactant, or a combination thereof. If theformulation is intended for in vivo administration, it is preferablethat the aqueous solution is a physiologically acceptable buffer thatmaintains a pH that is compatible with normal physiological conditions.Also, in certain instances, it may be desirable to maintain the pH at aparticular level in order to insure the stability of certain componentsof the formulation.

For example, it may be desirable to prepare an aqueous solution that isisotonic and/or isosmotic. Hypertonic and hypotonic solutions sometimescould cause complications and undesirable effects when injected, such aspost-administration swelling or rapid absorption of the compositionbecause of differential ion concentrations between the composition andphysiological fluids. To control tonicity, the emulsion may comprise aphysiological salt, such as a sodium salt. Sodium chloride (NaCl), forexample, may be used at about 0.9% (w/v) (physiological saline). Othersalts that may be present include potassium chloride, potassiumdihydrogen phosphate, disodium phosphate dehydrate, magnesium chloride,calcium chloride, etc. In an exemplary embodiment, the aqueous solutioncomprises 10 mM NaCl and other salts or non-ionic tonicifying agents. Asdescribed herein, non-ionic tonicifying agents can also be used tocontrol tonicity.

The aqueous solution may be buffered. Any physiologically acceptablebuffer may be used herein, such as citrate buffers, phosphate buffers,acetate buffers, succinate buffer, tris buffers, bicarbonate buffers,carbonate buffers, or the like. The pH of the aqueous solution willpreferably be between 6.0-8.0, more preferably about 6.2 to about 6.8.In some cases, certain amount of salt may be included in the buffer. Inother cases, salt in the buffer might interfere with complexation ofnegatively charged molecule to the emulsion particle, and therefore isavoided.

The aqueous solution may also comprise additional components such asmolecules that change the osmolarity of the aqueous solution ormolecules that stabilizes the negatively charged molecule aftercomplexation. For example, the osmolality can be adjusted using anon-ionic tonicifying agent, which are generally carbohydrates but canalso be polymers. (See, e.g., Voet and Voet (1990) Biochemistry (JohnWiley & Sons, New York.) Examples of suitable non-ionic tonicifyingagents include sugars (e.g., a monosaccharide, a disaccharide, or apolysaccharide, such as trehalose, sucrose, dextrose, fructose), sugaralcohols (e.g., mannitol, sorbitol, xylitol, erythritol, lactitol,maltitol, glycerol, reduced palatinose), and combinations thereof. Ifdesired, a nonionic polymer (e.g., a poly(alkyl glycol), such aspolyethylene glycol, polypropylene glycol, or polybutlyene glycol), ornonionic surfactant can be used. These types of agents, in particularsugar and sugar alcohols, are also cryoprotectants that can protect RNA,and other negatively charged molecules, when lyophilized. In exemplaryembodiments, the buffer comprises from about 560 nM to 600 mM oftrehalose, sucrose, sorbitol, or dextrose. In other exemplaryembodiments, the buffer comprises from about 500 nM to 600 mM oftrehalose, sucrose, sorbitol, or dextrose.

In some case, it may be preferable to prepare an aqueous solutioncomprising the negatively charged molecule as a hypertonic solution, andto prepare the cationic emulsion using unadulterated water or ahypotonic buffer. When the emulsion and the negatively charged moleculeare combined, the mixture becomes isotonic. For example, an aqueoussolution comprising RNA can be a 2× hypertonic solution, and thecationic emulsion can be prepared using 10 mM Citrate buffer. When theRNA solution and the emulsion are mixed at 1:1 (v/v) ratio, thecomposition becomes isotonic. Based on desired relative amounts of theemulsion to the aqueous solution that comprises the negatively chargedmolecule (e.g., 1:1 (v/v) mix, 2:1 (v/v) mix, 1:2 (v/v) mix, etc.), onecan readily determine the tonicity of the aqueous solution that isrequired in order to achieve an isotonic mixture.

Similarly, compositions that have physiological osmolality may bedesirable for in vivo administration. Physiological osmolality is fromabout 255 mOsm/kg water to about 315 mOsm/kg water. Sometimes, it may bepreferable to prepare an aqueous solution comprising the negativelycharged molecule as a hyperosmolar solution, and to prepare the cationicemulsion using unadulterated water or a hypoosmolar buffer. When theemulsion and the negatively charged molecule are combined, physiologicalosmolality is achieved. Based on desired relative amounts of theemulsion to the aqueous solution that comprises the negatively chargedmolecule (e.g., 1:1 (v/v) mix, 2:1 (v/v) mix, 1:2 (v/v) mix, etc.), onecan readily determine the osmolality of the aqueous solution that isrequired in order to achieve an iso-osmolar mixture.

In certain embodiments, the aqueous solution comprising the negativelycharged molecule may further comprise a polymer or a surfactant, or acombination thereof. In an exemplary embodiment, the oil-in-wateremulsion contains a poloxamer. In particular, the inventors haveobserved that adding Pluronic® F127 to the RNA aqueous solution prior tocomplexation to cationic emulsion particles led to greater stability andincreased RNase resistance of the RNA molecule. Addition of pluronicF127 to RNA aqueous solution was also found to decrease the particlesize of the RNA/CNE complex. Poloxamer polymers may also facilitateappropriate decomplexation/release of the RNA molecule, preventaggregation of the emulsion particles, and have immune modulatoryeffect. Other polymers that may be used include, e.g., Pluronic® F68 orPEG300.

Alternatively or in addition, the aqueous solution comprising thenegatively charged molecule may comprise from about 0.05% to about 20%(w/v) polymer. For example, the cationic oil-in-water emulsion maycomprise a polymer (e.g., a poloxamer such as Pluronic® F127, Pluronic®F68, or PEG300) at from about 0.05% to about 10% (w/v), such as 0.05%,0.5%, 1%, or 5%.

The buffer system may comprise any combination of two or more moleculesdescribed above (salt, buffer, saccharide, polymer, etc). In anpreferred embodiment, the buffer comprises 560 mM sucrose, 20 mM NaCl,and 2 mM Citrate, which can be mixed with a cationic oil in wateremulsion described herein to produce a final aqueous phase thatcomprises 280 mM sucrose, 10 mM NaCl and 1 mM citrate.

5. Methods of Preparation

In another aspect, the invention provides a method of preparing theoil-in-water emulsions as described herein, comprising: (1) combiningthe oil and the cationic lipid to form the oil phase of the emulsion;(2) providing an aqueous solution to form the aqueous phase of theemulsion; and (3) dispersing the oil phase in the aqueous phase, forexample, by homogenization. Homogenization may be achieved in anysuitable way, for example, using a commercial homogenizer (e.g., IKA T25homogenizer, available at VWR International (West Chester, Pa.).

In certain embodiments, the oil-in-water emulsions are prepared by (1)directly dissolving the cationic lipid in the oil to form an oil phase;(2) providing the aqueous phase of the emulsion; and (3) dispersing theoil phase in the aqueous phase by homogenization. The method does notuse an organic solvent (such as chloroform (CHCl₃), dichloromethane(DCM), ethanol, acetone, Tetrahydrofuran (THF), 2,2,2 trifluoroethanol,acetonitrile, ethyl acetate, hexane, Dimethylformamide (DMF), Dimethylsulfoxide (DMSO), etc.) to solubilize the cationic lipid first beforeadding the lipid to the oil.

It may be desirable to heat the oil to a temperature between about 37°C. to about 65° C. to facilitate the dissolving of the lipid. Desiredamount of the cationic lipid (e.g., DOTAP) can be measured and addeddirectly to the oil to reach a desired final concentration.

If the emulsion comprises one or more surfactants, the surfactant(s) maybe included in the oil phase or the aqueous phase according to theconventional practice in the art. For example, SPAN85 can be dissolvedin the oil phase (e.g., squalene), and Tween 80 may be dissolved in theaqueous phase (e.g., in a citrate buffer).

In another aspect, the invention provides a method of preparing acomposition that comprises a negatively charged molecule (such as RNA)complexed with a particle of a cationic oil-in-water emulsion,comprising: (i) providing a cationic oil-in-water emulsion as describedherein; (ii) providing a aqueous solution comprising the negativelycharged molecule (such as RNA); and (iii) combining the oil-in-wateremulsion of (i) and the aqueous solution of (iii), so that thenegatively charged molecule complexes with the particle of the emulsion.

For example, a cationic oil-in-water emulsion may be combined with anaqueous RNA solution in any desired relative amounts, e.g., about 1:1(v/v), about 1.5:1 (v/v), about 2:1 (v/v), about 2.5:1 (v/v), about 3:1(v/v), about 3.5:1 (v/v), about 4:1 (v/v), about 5:1 (v/v), about 10:1(v/v), about 1:1.5 (v/v), about 1:2 (v/v), about 1:2.5 (v/v), about 1:3(v/v), about 1:3.5 (v/v), about 1:4 (v/v), about 1:1.5 (v/v), or about1:1.10 (v/v), etc.

Additional optional steps to promote particle formation, to improve thecomplexation between the negatively charge molecules and the cationicparticles, to increase the stability of the negatively charge molecule(e.g., to prevent degradation of an RNA molecule), to facilitateappropriate decomplexation/release of the negatively charged molecules(such as an RNA molecule), or to prevent aggregation of the emulsionparticles may be included. For example, a polymer (e.g., Pluronic® F127)or a surfactant may be added to the aqueous solution that comprises thenegatively charged molecule (such as RNA).

The size of the emulsion particles can be varied by changing the ratioof surfactant to oil (increasing the ratio decreases particle size),operating pressure (increasing operating pressure reduces particlesize), temperature (increasing temperature decreases particle size), andother process parameters. Actual particle size will also vary with theparticular surfactant, oil, and cationic lipid used, and with theparticular operating conditions selected. Emulsion particle size can beverified by use of sizing instruments, such as the commercial Sub-MicronParticle Analyzer (Model N4MD) manufactured by the Coulter Corporation,and the parameters can be varied using the guidelines set forth aboveuntil the average diameter of the particles is less than less than about200 nm, less than about 150 nm, or less than about 100 nm. Preferably,the particles have an average diameter of about 180 nm or less, about150 nm or less, about 140 nm or less, or about 130 nm or less, about 120nm or less, or about 100 nm or less, from about 50 nm to 200 nm, fromabout 80 nm to 200 nm, from about 50 nm to 180 nm, from about 60 nm to180 nm, from about 70 to 180 nm, or from about 80 nm to 180 nm, fromabout 80 nm to about 170 nm, from about 80 nm to about 160 nm, fromabout 80 nm to about 150 nm, from about 80 nm to about 140 nm, fromabout 80 nm to about 130 nm, from about 80 nm to about 120 nm, fromabout 80 nm to about 110 nm, or from about 80 nm to about 100 nm.Emulsions wherein the mean particle size is about 200 nm or less allowfor sterile filtration.

Optional processes for preparing the cationic oil-in-water emulsion(pre-complexation emulsion), or the negatively charged molecule-emulsioncomplex, include, e.g., sterilization, particle size selection (e.g.,removing large particles), filling, packaging, and labeling, etc. Forexample, if the pre-complexation emulsion, or the negatively chargedmolecule-emulsion complex, is formulated for in vivo administration, itmay be sterilized. For example, the formulation can be sterilized byfiltering through a sterilizing grade filter (e.g., through a 0.22micron filter). Other sterilization techniques include a thermalprocess, or a radiation sterilization process, or using pulsed light toproduce a sterile composition.

The cationic oil-in-water emulsion described herein can be used tomanufacture vaccines. Sterile and/or clinical grade cationicoil-in-water emulsions can be prepared using similar methods asdescribed for MF59. See, e.g., Ott et al., Methods in MolecularMedicine, 2000, Volume 42, 211-228, in VACCINE ADJUVANTS (O'Hagan ed.),Humana Press. For example, similar to the manufacturing process of MF59,the oil phase and the aqueous phase of the emulsion can be combined andprocessed in a rotor stator homogenizer, or an inline homogenizer, toyield a coarse emulsion. The coarse emulsion can then be fed into amicrofluidizer, where it can be further processed to obtain a stablesubmicron emulsion. The coarse emulsion can be passed through theinteraction chamber of the microfluidizer repeatedly until the desiredparticle size is obtained. The bulk emulsion can then be filtered (e.g.,though a 0.22-μm filter under nitrogen) to remove large particles,yielding emulsion bulk that can be filled into suitable containers(e.g., glass bottles). For vaccine antigens that have demonstratedlong-term stability in the presence of oil-in-water emulsion for selfstorage, the antigen and emulsion may be combined and sterile-filtered(e.g., though a 0.22-μm filter membrane). The combined single vialvaccine can be filled into single-dose containers. For vaccine antigenswhere long-term stability has not been demonstrated, the emulsion can besupplied as a separate vial. In such cases, the emulsion bulk can befiltered-sterilized (e.g., though a 0.22-μm filter membrane), filled,and packaged in final single-dose vials.

Quality control may be optionally performed on a small sample of theemulsion bulk or admixed vaccine, and the bulk or admixed vaccine willbe packaged into doses only if the sample passes the quality controltest.

6. Kits, Pharmaceutical Compositions and Administration

In another aspect, the invention provides a pharmaceutical compositioncomprising a negatively charged molecule (such as RNA) complexed with aparticle of a cationic oil-in-water emulsion, as described herein, andmay further comprise one or more pharmaceutically acceptable carriers,diluents, or excipients. In preferred embodiments, the pharmaceuticalcomposition is an immunogenic composition, which can be used as avaccine.

Alternatively, the compositions described herein may be used to delivera negatively charged molecule to cells. For example, nucleic acidmolecules (e.g., DNA or RNA) can be delivered to cells for a variety ofpurposes, such as to induce production of a desired gene product (e.g.,protein), to regulate expression of a gene, for gene therapy and thelike. The compositions described herein may also be used to deliver anucleic acid molecule (e.g., DNA or RNA) to cells for therapeuticpurposes, such as to treat a disease such as cancers or proliferativedisorders, metabolic diseases, cardiovascular diseases, infections,allergies, to induce an immune response and the like. For example,nucleic acid molecules may be delivered to cells to inhibit theexpression of a target gene. Such nucleic acid molecules include, e.g.,antisense oligonucleotides, double-stranded RNAs, such as smallinterfering RNAs and the like. Double-stranded RNA molecules, such assmall interfering RNAs, can trigger RNA interference, which specificallysilences the corresponding target gene (gene knock down). Antisenseoligonucleotides are single strands of DNA or RNA that are complementaryto a chosen sequence. Generally, antisense RNA can prevent proteintranslation of certain messenger RNA strands by binding to them.Antisense DNA can be used to target a specific, complementary (coding ornon-coding) RNA. Therefore, the cationic emulsions described herein areuseful for delivering antisense oligonucleotides or double-stranded RNAsfor treatment of, for example, cancer by inhibiting production of anoncology target.

The invention also provides kits, wherein the negatively chargedmolecule (such as RNA) and the cationic oil-in-water emulsion are inseparate containers. For example, the kit can contain a first containercomprising a composition comprising the negatively charged molecule(such as RNA), and a second container comprising cationic oil-in-wateremulsion. The two components may be mixed prior to administration, e.g.,within about 72 hours, about 48 hours, about 24 hours, about 12 hours,about 10 hours, about 9 hours, about 8 hours, about 7 hours, about 6hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about1 hour, about 45 minutes, about 30 minutes, about 15 minutes, about 10minutes, about 5 minutes prior to administration. The two components mayalso be mixed about 1 minute or immediately prior to administration.

The negatively charged molecule (e.g., RNA) may be in liquid form or canbe in solid form (e.g., lyophilized). If in solid form, the kit maycomprise a third container comprising a suitable aqueous solution torehydrate the negatively charged molecule. Suitable aqueous solutionsinclude pharmaceutically-acceptable buffers such as phosphate-bufferedsaline, Ringer's solution, dextrose solution, or any one of the aqueoussolutions described above. In certain embodiments, sterile water may beused as the aqueous solution for rehydration, in particular in caseswhere additional components, such as tonicifying agents and/orosmolality adjusting agents are lyophilized along with the negativelycharged molecule (e.g., RNA). Alternatively, the lyophilized negativelycharged molecule (e.g., RNA) may be mixed directly with the cationicemulsion.

If the composition (e.g., a vaccine) comprises a negatively chargedmolecule (e.g., RNA) and an additional component, such as a proteinimmunogen, both components can be frozen and lyophilized (eitherseparately, or as a mixture), and reconstituted and mixed with thecationic emulsion prior to administration.

The kit can further comprise other materials useful to the end-user,including other pharmaceutically acceptable formulating solutions suchas buffers, diluents, filters, needles, and syringes or other deliverydevice. For example, the kit may include a dual chamber syringe thatcontain water or the emulsion in one chamber, and the negatively chargedmolecule (e.g., RNA) is provided in solid (e.g. lyophilized) form in theother chamber.

The kit may further include another container comprising an adjuvant(such as an aluminum containing adjuvant or MF59). In general, aluminumcontaining adjuvants are not preferred because they may interfere withthe complexation of the negatively charged molecule with the cationicemulsion.

Suitable containers for the compositions include, for example, bottles,vials, syringes, and test tubes. Containers can be formed from a varietyof materials, including glass or plastic. A container may have a sterileaccess port (for example, the container may be an intravenous solutionbag or a vial having a stopper pierceable by a hypodermic injectionneedle). Dual-chamber syringe may also be used, wherein the negativelycharged molecule (e.g., RNA) is lyophilized, and either reconstitutedwith water in the syringe, or reconstituted directly with a cationicemulsion described herein.

The kit can also comprise a package insert containing writteninstructions for methods of inducing immunity or for treatinginfections. The package insert can be an unapproved draft package insertor can be a package insert approved by the Food and Drug Administration(FDA) or other regulatory body.

The invention also provides a delivery device pre-filled with thecompositions described above.

The pharmaceutical compositions provided herein may be administeredsingly or in combination with one or more additional therapeutic agents.The method of administration include, but are not limited to, oraladministration, rectal administration, parenteral administration,subcutaneous administration, intravenous administration, intravitrealadministration, intramuscular administration, inhalation, intranasaladministration, topical administration, ophthalmic administration, orotic administration.

A therapeutically effective amount of the compositions described hereinwill vary depending on, among others, the disease indicated, theseverity of the disease, the age and relative health of the subject, thepotency of the compound administered, the mode of administration and thetreatment desired.

In other embodiments, the pharmaceutical compositions described hereincan be administered in combination with one or more additionaltherapeutic agents. The additional therapeutic agents may include, butare not limited to antibiotics or antibacterial agents, antiemeticagents, antifungal agents, anti-inflammatory agents, antiviral agents,immunomodulatory agents, cytokines, antidepressants, hormones,alkylating agents, antimetabolites, antitumour antibiotics, antimitoticagents, topoisomerase inhibitors, cytostatic agents, anti-invasionagents, antiangiogenic agents, inhibitors of growth factor functioninhibitors of viral replication, viral enzyme inhibitors, anticanceragents, α-interferons, β-interferons, ribavirin, hormones, and othertoll-like receptor modulators, immunoglobulins (Igs), and antibodiesmodulating Ig function (such as anti-IgE (omalizumab)).

In certain embodiments, the pharmaceutical compositions provided hereinare used in the treatment of infectious diseases including, but notlimited to, disease cased by the pathogens disclosed herein, includingviral diseases such as genital warts, common warts, plantar warts,rabies, respiratory syncytial virus (RSV), hepatitis B, hepatitis C,Dengue virus, yellow fever, herpes simplex virus (by way of exampleonly, HSV-I, HSV-II, CMV, or VZV), molluscum contagiosum, vaccinia,variola, lentivirus, human immunodeficiency virus (HIV), human papillomavirus (HPV), hepatitis virus (hepatitis C virus, hepatitis B virus,hepatitis A virus), cytomegalovirus (CMV), varicella zoster virus (VZV),rhinovirus, enterovirus (e.g. EV71), adenovirus, coronavirus (e.g.,SARS), influenza, para-influenza, mumps virus, measles virus, rubellavirus, papovavirus, hepadnavirus, flavivirus, retrovirus, arenavirus (byway of example only, LCM, Junin virus, Machupo virus, Guanarito virusand Lassa Fever) and filovirus (by way of example only, ebola virus ormarburg virus).

In certain embodiments, the pharmaceutical compositions provided hereinare used in the treatment of bacterial, fungal, and protozoal infectionsincluding, but not limited to, malaria, tuberculosis and mycobacteriumavium, leprosy; pneumocystis carnii, cryptosporidiosis, histoplasmosis,toxoplasmosis, trypanosome infection, leishmaniasis, infections causedby bacteria of the genus Escherichia, Enterobacter, Salmonella,Staphylococcus, Klebsiella, Proteus, Pseudomonas, Streptococcus, andChlamydia, and fungal infections such as candidiasis, aspergillosis,histoplasmosis, and cryptococcal meningitis.

In certain embodiments, the pharmaceutical compositions provided hereinare used in the treatment of respiratory diseases and/or disorders,dermatological disorders, ocular diseases and/or disorders,genitourinary diseases and/or disorders including, allograft rejection,auto-immune and allergic, cancer, or damaged or ageing skin such asscarring and wrinkles.

In another aspect, the invention provides a method for generating orpotentiating an immune response in a subject in need thereof, such as amammal, comprising administering an effective amount of a composition asdisclosed herein. The immune response is preferably protective andpreferably involves antibodies and/or cell-mediated immunity. The methodmay be used to induce a primary immune response and/or to boost animmune response.

In certain embodiments, the compositions disclosed herein may be used asa medicament, e.g., for use in raising or enhancing an immune responsein a subject in need thereof, such as a mammal.

In certain embodiments, the compositions disclosed herein may be used inthe manufacture of a medicament for generating or potentiating an immuneresponse in a subject in need thereof, such as a mammal.

The mammal is preferably a human, but may be, e.g., a cow, a pig, achicken, a cat or a dog, as the pathogens covered herein may beproblematic across a wide range of species. Where the vaccine is forprophylactic use, the human is preferably a child (e.g., a toddler orinfant), a teenager, or an adult; where the vaccine is for therapeuticuse, the human is preferably a teenager or an adult. A vaccine intendedfor children may also be administered to adults, e.g., to assess safety,dosage, immunogenicity, etc.

One way of checking efficacy of therapeutic treatment involvesmonitoring pathogen infection after administration of the compositionsor vaccines disclosed herein. One way of checking efficacy ofprophylactic treatment involves monitoring immune responses,systemically (such as monitoring the level of IgG1 and IgG2a production)and/or mucosally (such as monitoring the level of IgA production),against the antigen. Typically, antigen-specific serum antibodyresponses are determined post-immunization but pre-challenge whereasantigen-specific mucosal antibody responses are determinedpost-immunization and post-challenge.

Another way of assessing the immunogenicity of the compositions orvaccines disclosed herein where the nucleic acid molecule (e.g., theRNA) encodes a protein antigen is to express the protein antigenrecombinantly for screening patient sera or mucosal secretions byimmunoblot and/or microarrays. A positive reaction between the proteinand the patient sample indicates that the patient has mounted an immuneresponse to the protein in question. This method may also be used toidentify immunodominant antigens and/or epitopes within proteinantigens.

The efficacy of the compositions can also be determined in vivo bychallenging appropriate animal models of the pathogen of interestinfection.

Dosage can be by a single dose schedule or a multiple dose schedule.Multiple doses may be used in a primary immunization schedule and/or ina booster immunization schedule. In a multiple dose schedule the variousdoses may be given by the same or different routes, e.g., a parenteralprime and mucosal boost, a mucosal prime and parenteral boost, etc.Multiple doses will typically be administered at least 1 week apart(e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.).

In certain embodiments, the total amount of cationic lipid, such asDOTAP, that is administered to the subject in a single administration isno more than about 30 mg, or no more than about 24 mg.

In certain embodiments, the total amount of cationic lipid, such asDOTAP, that is administered to the subject in a single administration isno more than 4 mg.

The compositions disclosed herein that include one or more antigens orare used in conjunction with one or more antigens may be used to treatboth children and adults. Thus a human subject may be less than 1 yearold, 1-5 years old, 5-15 years old, 15-55 years old, or at least 55years old. Preferred subjects for receiving the compositions are theelderly (e.g., >50 years old, >60 years old, and preferably >65 years),the young (e.g., <5 years old), hospitalized patients, healthcareworkers, armed service and military personnel, pregnant women, thechronically ill, or immunodeficient patients. The compositions are notsuitable solely for these groups, however, and may be used moregenerally in a population.

The compositions disclosed herein that include one or more antigens orare used in conjunction with one or more antigens may be administered topatients at substantially the same time as (e.g., during the samemedical consultation or visit to a healthcare professional orvaccination centre) other vaccines, e.g., at substantially the same timeas a measles vaccine, a mumps vaccine, a rubella vaccine, a MMR vaccine,a varicella vaccine, a MMRV vaccine, a diphtheria vaccine, a tetanusvaccine, a pertussis vaccine, a DTP vaccine, a conjugated H. influenzaetype b vaccine, an inactivated poliovirus vaccine, a hepatitis B virusvaccine, a meningococcal conjugate vaccine (such as a tetravalent A CW135 Y vaccine), a respiratory syncytial virus vaccine, etc.

In certain embodiments, the compositions provided herein include oroptionally include one or more immunoregulatory agents such asadjuvants. Exemplary adjuvants include, but are not limited to, a TH1adjuvant and/or a TH2 adjuvant, further discussed below. In certainembodiments, the adjuvants used in the immunogenic compositions provideherein include, but are not limited to:

-   -   1. Mineral-Containing Compositions;    -   2. Oil Emulsions;    -   3. Saponin Formulations;    -   4. Virosomes and Virus-Like Particles;    -   5. Bacterial or Microbial Derivatives;    -   6. Bioadhesives and Mucoadhesives;    -   7. Liposomes;    -   8. Polyoxyethylene Ether and Polyoxyethylene Ester Formulations;    -   9. Polyphosphazene (PCPP);    -   10. Muramyl Peptides;    -   11. Imidazoquinolone Compounds;    -   12. Thiosemicarbazone Compounds;    -   13. Tryptanthrin Compounds;    -   14. Human Immunomodulators;    -   15. Lipopeptides;    -   16. Benzonaphthyridines;    -   17. Microparticles    -   18. Immunostimulatory polynucleotide (such as RNA or DNA; e.g.,        CpG-containing oligonucleotides)

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1: Development of Cationic Oil-in-Water Emulsions

In this Example, cationic nanoemulsions (referred herein as “CNEs”) thatcontain high concentrations of cationic lipid (DOTAP) were developed forthe delivery of self replicating RNA.

The CNE formulations are summarized in Table 1 below, and were modifiedbased on CNE01. CNE01, CMF40, CNE16, CNE02, and CNE17 were used asreference samples for comparative studies.

TABLE 1 oil:Lipid Cationic ratio Aqueous CNE Lipid mg/mL SurfactantSqualene (mole:mole) phase Ref. 1 CNE01 DOTAP 0.5% SPAN 85  4.3% 91.7:110 mM (in CHCl₃) 0.5% Tween 80 citrate buffer 0.8 pH 6.5 Ref. 2 CMF40DOTAP 0.5% SPAN 85  4.3% 73.3:1 10 mM (no organic 0.5% Tween 80 citratebuffer solvent) pH 6.5 1.0 Ref. 3 CNE16 DOTAP 0.5% SPAN 85  4.3% 61.1:110 mM (no organic 0.5% Tween 80 citrate buffer solvent) pH 6.5 1.2 Ref.4 CNE02 DOTAP 0.5% SPAN 85  4.3% 45.8:1 10 mM (no organic 0.5% Tween 80citrate buffer solvent) pH 6.5 1.6 Ref. 5 CNE17 DOTAP 0.5% SPAN 85  4.3%52.4:1 10 mM (in DCM) 0.5% Tween 80 citrate buffer 1.4 pH 6.5 ExampleCMF41 DOTAP 0.5% SPAN 85  4.3% 40.7:1 10 mM 1 (no organic 0.5% Tween 80citrate buffer solvent) pH 6.5 1.8 Example CMF30 DOTAP 0.5% SPAN 85 4.3% 36.7:1 10 mM 2 (no organic 0.5% Tween 80 citrate buffer solvent)pH 6.5 2.0 Example CMF31 DOTAP 0.5% SPAN 85  4.3% 28.2:1 10 mM 3 (noorganic 0.5% Tween 80 citrate buffer solvent) pH 6.5 2.6 Example CMF32DOTAP 0.5% SPAN 85  4.3% 22.9:1 10 mM 4 (no organic 0.5% Tween 80citrate buffer solvent) pH 6.5 3.2 Example CMF33 DOTAP 0.5% SPAN 85 4.3% 19.3:1 10 mM 5 (no organic 0.5% Tween 80 citrate buffer solvent)pH 6.5 3.8 Example CMF34 DOTAP 0.5% SPAN 85  4.3% 16.7:1 10 mM 6 (noorganic 0.5% Tween 80 citrate buffer solvent) pH 6.5 4.4 Example CMF35DOTAP 0.5% SPAN 85  4.3% 14.7:1 10 mM 7 (no organic 0.5% Tween 80citrate buffer solvent) pH 6.5 5.0 Example CMF44 DOTAP 0.5% SPAN 853.23% 12.5:1 10 mM 8 (no organic 0.5% Tween 80 citrate buffer solvent)pH 6.5 4.4 Example CMF45 DOTAP 0.5% SPAN 85 2.15%  8.4:1 10 mM 9 (noorganic 0.5% Tween 80 citrate buffer solvent) pH 6.5 4.4 Example CMF46DOTAP 0.5% SPAN 85 1.08%  4.2:1 10 mM 10  (no organic 0.5% Tween 80citrate buffer solvent) pH 6.5 4.4

CNEs were prepared similar to charged MF59 as previously described (Ottet al., Journal of Controlled Release, volume 79, pages 1-5, 2002), withone major modification. DOTAP was dissolved in the squalene directly,and no organic solvent was used. It was discovered that inclusion of asolvent in emulsions that contained greater than 1.6 mg/ml DOTAPproduced a foamy feedstock that could not be microfluidized to producean emulsion. Heating squalene to 37° C. allowed DOTAP to be directlydissolved in squalene, and then the oil phase could be successfullydispersed in the aqueous phase (e.g., by homogenization) to produce anemulsion. DOTAP is soluble in squalene and higher concentrations ofDOTAP in squalene than those listed in Table 1 may be achieved. However,it has been reported that high dose of DOTAP can have toxic effects.See, e.g., Lappalainen et al., Pharm. Res., vol. 11(8):1127-31 (1994).

Briefly, squalene was heated to 37° C., and DOTAP was dissolved directlyin squalene in the presence of SPAN 85. The resulting oil phase was thencombined with the aqueous phase (Tween 80 in citrate buffer) andimmediately homogenized for 2 min using an IKA T25 homogenizer at 24KRPM to produce a homogeneous feedstock (primary emulsions). The primaryemulsions were passed three to five times through a M-110SMicrofluidizer or a M-110P Microfluidizer (Microfluidics, Newton, Mass.)with an ice bath cooling coil at a homogenization pressure ofapproximately 15K-20K PSI. The 20 ml batch samples were removed from theunit and stored at 4° C.

It should be noted that the concentrations of the components of theCNEs, as describes in Table 1, are concentrations calculated accordingthe initial amounts of these components that were used to prepare theemulsions. It is understood that during the process of producingemulsions, or during the filter sterilization process, small amounts ofsqualene, DOTAP, or other components may be lost, and the actualconcentrations of these components in the final product (e.g., apackaged, sterilized emulsion that is ready for administration) might beslightly lower, typically by up to about 20%, sometimes by up to about25%, or up to about 35%. However, the conventional practice in the artis to describe the concentration of a particular component based on theinitial amount that is used to prepare the emulsion, instead of theactual concentration in the final product.

Table 2 below shows the difference between the “theoretical”concentrations of squalene and DOTAP (calculated according the initialamounts of squalene and DOTAP that were used to prepare the emulsions),and the actual concentrations of squalene and DOTAP as measured in thefinal product.

TABLE 2 % of % of Theoretical Actual Theoretical Theoretical ActualTheoretical DOTAP DOTAP DOTAP Squalene Squalene Squalene CNE (mg/mL)(mg/mL) Yield (mg/mL) (mg/mL) Yield CMF32 Batch 1 3.2 2.20 68.76 4319.33 44.95 CMF32 Batch 2 3.2 2.57 80.32 43 34.45 80.12 CMF32 Batch 33.2 2.37 73.95 43 38.38 89.25 CMF34 Batch 1 4.4 2.75 62.44 43 30.4670.84 CMF34 Batch 2 4.4 3.21 73.00 43 33.98 79.02 CMF34 Batch 3 4.4 3.0870.08 43 32.71 76.07 CMF34 Batch 4 4.4 3.52 79.93 43 28.95 67.34

Example 2: Preparation RNA-Particle Complexes

1. RNA Synthesis

Plasmid DNA encoding an alphavirus replicon (self-replicating RNA) wasused as a template for synthesis of RNA in vitro. Each replicon containsthe genetic elements required for RNA replication but lacks sequencesencoding gene products that are necessary for particle assembly. Thestructural genes of the alphavirus genome were replaced by sequencesencoding a heterologous protein (whose expression is driven by thealphavirus subgenomic promoter). Upon delivery of the replicons toeukaryotic cells, the positive-stranded RNA is translated to producefour non-structural proteins, which together replicate the genomic RNAand transcribe abundant subgenomic mRNAs encoding the heterologousprotein. Due to the lack of expression of the alphavirus structuralproteins, replicons are incapable of generating infectious particles. Abacteriophage T7 promoter is located upstream of the alphavirus cDNA tofacilitate the synthesis of the replicon RNA in vitro, and the hepatitisdelta virus (HDV) ribozyme located immediately downstream of thepoly(A)-tail generates the correct 3′-end through its self-cleavingactivity.

Following linearization of the plasmid DNA downstream of the HDVribozyme with a suitable restriction endonuclease, run-off transcriptswere synthesized in vitro using T7 or SP6 bacteriophage derivedDNA-dependent RNA polymerase. Transcriptions were performed for 2 hoursat 37° C. in the presence of 7.5 mM (T7 RNA polymerase) or 5 mM (SP6 RNApolymerase) final concentration of each of the nucleoside triphosphates(ATP, CTP, GTP and UTP) following the instructions provided by themanufacturer (Ambion, Austin, Tex.). Following transcription, thetemplate DNA was digested with TURBO DNase (Ambion, Austin, Tex.). Thereplicon RNA was precipitated with LiCl and reconstituted innuclease-free water. Uncapped RNA was capped post-transcriptionally withVaccinia Capping Enzyme (VCE) using the ScriptCap m⁷G Capping System(Epicentre Biotechnologies, Madison, Wis.) as outlined in the usermanual. Post-transcriptionally capped RNA was precipitated with LiCl andreconstituted in nuclease-free water. Alternatively, replicons may becapped by supplementing the transcription reactions with 6 mM (for T7RNA polymerase) or 4 mM (for SP6 RNA polymerase) m⁷G(5′)ppp(5′)G, anonreversible cap structure analog (New England Biolabs, Beverly, Mass.)and lowering the concentration of guanosine triphosphate to 1.5 mM (forT7 RNA polymerase) or 1 mM (for SP6 RNA polymerase). The transcripts maybe then purified by TURBO DNase (Ambion, Austin, Tex.) digestionfollowed by LiCL precipitation and a wash in 75% ethanol.

The concentration of the RNA samples was determined by measuring theoptical density at 260 nm. Integrity of the in vitro transcripts wasconfirmed by denaturing agarose gel electrophoresis for the presence ofthe full length construct.

2. RNA Complexation

The term N/P ratio refers to the amount of nitrogen in the cationiclipid in relation to the amount of phosphates on the RNA. The nitrogenis the charge bearing element within the cationic lipids tested. Thephosphate can be found on the RNA backbone. An N/P charge ratio of 10/1indicates that there are 10 positively charged nitrogen from thecationic lipid present for each negatively charged phosphate on the RNA.

The number of nitrogens in solution was calculated from the cationiclipid concentration, DOTAP for example has one nitrogen that can beprotonated per molecule. The RNA concentration was used to calculate theamount of phosphate in solution using an estimate of 3 nmols ofphosphate per microgram of RNA. By varying the amount of RNA:Lipid, theN/P ratio can be modified. RNA was complexed to the CNEs in a range ofnitrogen/phosphate ratios (N/P). Calculation of the N/P ratio was doneby calculating the number of moles of protonatable nitrogens in theemulsion per milliliter. To calculate the number of phosphates, aconstant of 3 nmols of phosphate per microgram of RNA was used. Afterthe values were determined, the appropriate ratio of the emulsion wasadded to the RNA. Using these values, the RNA was diluted to theappropriate concentration and added directly into an equal volume ofemulsion while vortexing lightly. The solution was allowed to sit atroom temperature for approximately 2 hours. Once complexed the resultingsolution was diluted to the appropriate concentration and used within 1hour.

3. Particle Size Assay

Particle size of the emulsion was measured using a Zetasizer Nano ZS(Malvern Instruments, Worcestershire, UK) according to themanufacturer's instructions. Particle sizes are reported as theZ-Average (ZAve) with the polydispersity index (pdi). All samples werediluted in water prior to measurements. Additionally, particle size ofthe emulsion was measured using Horiba LA-930 particle sizer (HoribaScientific, USA). Samples were diluted in water prior to measurements.Zeta potential was measured using Zetasizer Nano ZS using dilutedsamples according to the manufacturer's instructions.

4. Viral Replicon Particles (VRP)

To compare RNA vaccines to traditional RNA-vectored approaches forachieving in vivo expression of reporter genes or antigens, we utilizedviral replicon particles (VRPs) produced in BHK cells by the methodsdescribed by Perri et al., J. Virol, 77:10394-10403 (2003). In thissystem, the antigen (or reporter gene) replicons consisted of alphaviruschimeric replicons (VCR) derived from the genome of Venezuelan equineencephalitis virus (VEEV) engineered to contain the 3′ terminalsequences (3′ UTR) of Sindbis virus and a Sindbis virus packaging signal(PS) (see FIG. 2 of Perri S., et al., J Virol 77: 10394-10403 (2003)).These replicons were packaged into VRPs by co-electroporating them intobaby hamster kidney (BHK) cells along with defective helper RNAsencoding the Sindbis virus capsid and glycoprotein genes (see FIG. 2 ofPerri et al). The VRPs were then harvested and titrated by standardmethods and inoculated into animals in culture fluid or other isotonicbuffers.

Example 3: The Effect of DOTAP Concentration on Immunogenicity

This Example shows that cationic oil-in-water emulsions made with highconcentrations of DOTAP increased the immunogenicity of an RNA repliconthat encodes the RSV-F antigen in a mouse model.

1. Materials and Methods

Heparin Binding Assay

RNA was complexed as described above. The RNA/CNE complex was incubatedwith various concentrations of heparin sulfate (Alfa Aesar, Ward HillMass.) for 30 minutes at Room Temperature. The resulting solutions werethen placed on an Airfuge high speed centrifuge (Beckman Coulter, Brea,Calif.) for 15 minutes. The centrifuge tubes were punctured with atuberculin syringe and the subnatant was removed. The solution was thenassayed for RNA concentration using the Ribogreen assay (Invitrogen,Carlsbad Calif.) according to the manufactures directions. The sampleswere analyzed on a Biotek Synergy 4 (Winooski, Vt.) fluorescent platereader. Free RNA values were calculated using a standard curve.

2. The Effect of DOTAP Concentration on RNA-Particle Interactions

Table 3 shows the effect of DOTAP concentration on RNA-particleinteractions (as determined by Heparin binding assay, which measured thetightness of the RNA-particle interactions) and immunogenicity.

TABLE 3 Heparin Binding Assay DOTAP % of RNA release concentration in 8Xheparin CNE (mg/mL) N/P ratio Sulfate CNE01 0.8 2:1 nt 4:1 nt 6:1 62.828:1 54.18 10:1  nt 12:1  116.6 14:1  62.79 CMF41 1.0 2:1 nt 4:1 4.61 6:133.41 8:1 70.68 10:1  54.92 12:1  52.93 CNE16 1.2 2:1 nt 4:1 1.83 6:1 nt8:1 33.79 10:1  58.86 12:1  68.02 14:1  55.07 CNE17 1.4 2:1 nt 4:1 nt6:1 3.91 8:1 44.00 10:1  69.65 12:1  61.53 14:1  57.26 CNE02 1.6 2:1 nt4:1 nt 6:1 2.01 8:1 2.87 10:1  7.38 12:1  19.37 14:1  21.44 CMF41 1.82:1 nt 4:1 0.76 6:1 1.33 8:1 1.10 10:1  2.69 12:1  2.59 14:1  3.67 CMF302.0 2:1 nt 4:1 0.7 6:1 0.81 8:1 1.17 10:1  2.35 12:1  5.15 14:1  9.44CMF30 2.6 2:1 nt 4:1 nt 6:1 0.83 8:1 1.18 10:1  1.00 12:1  0.96 14:1 1.10 nt = not tested.

As shown in Table 3, RNA molecules bound strongly to emulsion particlesthat were made with high concentrations of DOTAP (1.8 mg/mL or higher).

3. The Effect of DOTAP Concentration on RNA Loading

Table 4 shows the effect of DOTAP concentration on RNA loading.Increasing the concentration of DOTAP resulted in higher amount of RNAmolecules being formulated into RNA-particle complexes.

TABLE 4 CNE CNE17 CMF41 CMF30 CMF31 CMF32 CMF33 CMF34 CMF35 DOTAP (in0.5 ml emulsion) 0.35 mg 0.45 mg 0.5 mg 0.65 mg 0.8 mg 0.95 mg 1.1 mg1.25 mg N/P ratio Amount of RNA (μg)  4 to 1 41.8 53.7 59.6 77.5 95.4113.3 131.2 149.1  6 to 1 27.8 35.8 39.8 51.7 63.6 75.6 87.5 99.4  8 to1 20.9 26.8 29.8 38.8 47.7 56.7 65.6 74.6 10 to 1 16.7 21.5 23.9 31 38.245.3 52.5 59.6 12 to 1 13.9 17.9 19.9 25.8 31.8 37.8 43.7 49.7 14 to 111.9 15.3 17 22.2 27.3 32.4 37.5 42.6

4. The Effect of DOTAP Concentration on Immunogenicity

Table 5 shows the effect of DOTAP concentration on the immunogenicity ofthe RSV F antigen in an in vivo mouse model.

The vA317 replicon that expresses the surface fusion glycoprotein of RSV(RSV-F) was used for this study. BALB/c mice, aged 8-10 weeks andweighing about 20 g, 10 animals per group, were given bilateralintramuscular vaccinations. All animals were injected in the quadricepsin the two hind legs each getting an equivalent volume (50 μL per leg)on days 0 and 21 with naked self-replicating RNA expressing RSV-F(vA317, 1 μg), 1 μg of A317 formulated in a liposome that contained 40%DlinDMA, 10% DSPC, 48% Chol, 2% PEG DMG 2000 (RV01(15)), orself-replicating RNA formulated in the indicated CNEs (1 vA317). Foreach administration, the formulations were freshly prepared. Serum wascollected for antibody analysis on days 14 (2wp1) and 35 (2wp2).

TABLE 5 RNA DOTAP 2wp1 2wp2/ CNE (μg/ N/P (mg/ GMT 2wp2 2wp1 ([ ] DOTAP)0.5 mL) ratio 0.5 mL) (Pooled) GMT ratio 1 μg vA317 — — — 764 344 0.5 1μg RV01(15) — — — 3898 66348 17.0 CNE01 9.55 10:1 0.20 163 993 6.1 (0.8mg/mL) CMF40 11.93 10:1 0.25 505 3350 6.6 (1.0 mg/mL) CNE16 14.32 10:10.30 465 3851 8.3 (1.2 mg/mL) CNE17 16.70 10:1 0.35 843 3638 4.3 (1.4mg/mL) CNE02 19.09 10:1 0.40 1253 5507 4.4 (1.6 mg/mL) CMF41 21.48 10:10.45 961 5132 5.3 (1.8 mg/mL) CMF30 23.86 10:1 0.50 2021 10068 5.0 (2.0mg/mL) CMF31 31.02 10:1 0.65 1557 11940 7.7 (2.6 mg/mL) CMF32 38.18 10:10.80 1124 6941 6.2 (3.2 mg/mL)

As shown in Table 5, increasing DOTAP concentration resulted in higheramount of RNA being loaded to the emulsion particles, which in turnincreased the host immune response. A 3-fold increase in antibody titer(at 2wp2) for CMF31 was observed as compared to CNE17. In this model, aplateau in immunogenicity was observed at 2.6 mg/mL DOTAP (CMF31).

When the amounts of RNA and DOTAP administered to each mouse were heldconstant (meaning for emulsions with higher concentrations of DOTAP,smaller volumes of emulsion were used to prepare the RNA/emulsioncomplex; then, prior to immunization, the RNA/emulsion formulations werediluted such that the volumes of the RNA/emulsion formulations injectedto the mice were the same), F-specific total IgG titers were comparablewith different CNE formulations (Table 6). vA317 replicon was used forall CNE formulations. RNAs were made with Ambion kit. The GMT datareflect the geometric mean titer of individual mice in each group (8mice/group). The result shows that smaller amount of the formulationswere needed for emulsions with higher concentrations of DOTAP.

TABLE 6 % of 2wp2/ max geo RNA N/P DOTAP Squalene 2wp1 2wp2 2wp1 meantiter, Formulation (μg/dose) ratio (μg/dose) (mg/dose) GMT GMT (boost)2wp2 Naked RNA 1 — — — 764 334 0 0 RV01 particles 1 — — — 3898 66348 17— CNE17 1 10:1 21 0.65 673 5314 8 41 CMF41 1 10:1 21 0.50 784 7083 9 55CMF30 1 10:1 21 0.45 492 8543 17 66 CMF31 1 10:1 21 0.35 1123 6972 6 54CMF32 1 10:1 21 0.28 1665 10498 6 82 CMF33 1 10:1 21 0.24 1351 12279 996 CMF34 1 10:1 21 0.20 936 12851 14 100 CMF35 1 10:1 21 0.18 628 776612 60Titers from pre-immunization serum contained undetectable titers.

When the amount of squalene and N/P ratio (DOTAP:RNA) administered toeach mouse were held constant, F-specific total IgG titers increased asthe amount of RNA and DOTAP in the formulations increased (Table 7). ThevA317 replicon was used for all CNE formulations. RNAs were made withAmbion kit. The GMT data reflect the geometric mean titer of individualmice in each group (8 mice/group). The result shows that increasingDOTAP concentration resulted in higher amount of RNA being loaded to theemulsion particles, which in turn increased the host immune response.

TABLE 7 % of 2wp2/ max geo RNA N/P DOTAP Squalene 2wp1 2wp2 2wp1 meantiter, Formulation (μg/dose) ratio (μg/dose) (mg/dose) GMT GMT (boost)2wp2 Naked 11.9 — — — 14 682 49 2 RV01 particles 3.3 — — — 3767 64889 17— RV01 particles 11.9 — — — 6562 102359 16 — CNE17 0 — 70 2.15 5 5 1 1CMF35 0 — 250 2.15 10 5 1 1 CNE17 3.3 10:1 70 2.15 223 8567 38 25 CMF414.3 10:1 90 2.15 974 7020 7 21 CMF30 4.8 10:1 100 2.15 1212 10999 9 33CMF31 6.2 10:1 130 2.15 874 15142 17 45 CMF32 7.6 10:1 160 2.15 181622239 12 66 CMF33 9.1 10:1 190 2.15 1862 17445 9 52 CMF34 10.5 10:1 2202.15 1302 33634 26 100 CMF35 11.9 10:1 250 2.15 1554 24971 16 74 Naïve —— — — 5 5 1 0

CMF32 and CMF34 were further studied using different N/P ratios. Table 8shows the F-specific total IgG titers of the formulations. TheoreticalN/P ratios reflect the N/P ratios calculated according to the initialamounts of DOTAP and RNA that were used to prepare the formulations.Actual N/P ratios were slightly lower than theoretical N/P ratiosbecause small amounts of DOTAP were lost during preparation of theemulsions. The vA317 was used for all CNE and CMF formulations. The GMTdata reflect the mean log₁₀ titer of individual mice in each group (8mice/group). All formulations were adjusted to 300 mOsm/kg with sucrose.There were no obvious tolerability issues observed (e.g., body weight,early serum cytokines) with either CMF32 or CMF34 formulations.

Actual N/P ratios were determined by quantifying DOTAP content in CNE orCMF batches using HPLC with a charged aerosol detector (Corona Ultra,Chelmsford, Mass.). The CNE and CMF samples were diluted in isopropanoland injected onto a XTera C18 4.6×150 mm 3.5 um column (Waters, Milford,Mass.). The area under the curve was taken from the DOTAP peak in thechromatogram and the concentration was interpolated off a DOTAP standardcurve. Using the actual DOTAP concentration, an actual N/P ratio was becalculated.

TABLE 8 Actual 2wp2/ RNA Theoretical N/P 2wp1 2wp1 2wp1 Formulation(μg/dose) N/P ratio ratio GMT GMT (boost) Naked 1 — — 68 1019 15 RV01 1— — 9883 68116 7 CNE17 1 10:1 — 1496 6422 4 CMF32 1 12:1 9.4:1 261714246 5 1 10:1 (batch 1) 6.0:1 1537 10575 7 1 10:1 (batch 2) 8.0:1 204716244 8 1  8:1 6.3:1 2669 7656 3 1  6:1 4.7:1 1713 4715 3 1  4:1 3.1:1872 3773 4 CMF34 1 12:1 7.4:1 3141 10134 3 1 10:1 (batch 1) 6.1:1 190611081 6 1 10:1 (batch 2) 7.0:1 2388 9857 4 1  8:1   5:1 1913 8180 4 1 6:1 3.7:1 1764 6209 4 1  4:1 2.5:1 1148 4936 4

Example 4: The Effect of Dotap Concentration on Immunogenicity

This Example shows that cationic oil-in-water emulsions made with highconcentrations of DOTAP increased the immunogenicity of an RNA repliconthat encodes the RSV-F antigen in a cotton rat model.

1. Materials and Methods

RNA Replicon.

The sequence of the RNA replicon, vA142 RSV-F-delFP-full ribozyme

Vaccination of cotton rats. Female cotton rats (Sigmodon hispidis) wereobtained from Harlan Laboratories. All studies were approved andperformed according to Novartis Animal Care and Use Committee. Groups ofanimals were immunized intramuscularly (i.m., 100 μl) with the indicatedvaccines on day 0. Serum samples were collected 3 weeks after eachimmunization Immunized or unvaccinated control animals were challengedintranasally (i.n.) with 1×10⁵ PFU RSV 4 weeks after the finalimmunization.

RSV-F Trimer Subunit Vaccine.

The RSV F trimer is a recombinant protein comprising the ectodomain ofRSV F with a deletion of the fusion peptide region preventingassociation with other trimers. The resulting construct forms ahomogeneous trimer, as observed by size exclusion chromatography, andhas an expected phenotype consistent with a postfusion F conformation asobserved by electron microscopy. The protein was expressed in insectcells or CHO cells and purified by virtue of a HIS-tagged in fusion withthe construct's C-terminus followed by size exclusion chromatographyusing conventional techniques. The resulting protein sample exhibitsgreater than 95% purity. For the in vivo evaluation of the F-subunitvaccine, 100 μg/mL trimer protein was adsorbed on 2 mg/mL alum using 10mM Histidine buffer, pH 6.3 and isotonicity adjusted with sodiumchloride to 150 mM. F-subunit protein was adsorbed on alum overnightwith gentle stirring at 2-8° C.

RSV F-Specific ELISA.

Individual serum samples were assayed for the presence of RSV F-specificIgG by enzyme-linked immunosorbent assay (ELISA). ELISA plates (MaxiSorp96-well, Nunc) were coated overnight at 4° C. with 1 μg/ml purified RSVF (delp23-furdel-trunc uncleaved) in PBS. After washing (PBS with 0.1%Tween-20), plates were blocked with Superblock Blocking Buffer in PBS(Thermo Scientific) for at least 1.5 hr at 37° C. The plates were thenwashed, serial dilutions of serum in assay diluent (PBS with 0.1%Tween-20 and 5% goat serum) from experimental or control cotton ratswere added, and plates were incubated for 2 hr at 37° C. After washing,plates were incubated with horse radish peroxidase (HRP)-conjugatedchicken anti-cotton rat IgG (Immunology Consultants Laboratory, Inc,diluted 1:5,000 in assay diluent) for 1 hr at 37° C. Finally, plateswere washed and 100 μl of TMB peroxidase substrate solution (Kirkegaard& Perry Laboratories, Inc) was added to each well. Reactions werestopped by addition of 100 μl of 1M H₃PO₄, and absorbance was read at450 nm using a plate reader. For each serum sample, a plot of opticaldensity (OD) versus logarithm of the reciprocal serum dilution wasgenerated by nonlinear regression (GraphPad Prism). Titers were definedas the reciprocal serum dilution at an OD of approximately 0.5(normalized to a standard, pooled sera from RSV-infected cotton ratswith a defined titer of 1:2500, that was included on every plate).

Micro Neutralization Assay.

Serum samples were tested for the presence of neutralizing antibodies bya plaque reduction neutralization test (PRNT). Two-fold serial dilutionsof HI-serum (in PBS with 5% HI-FBS) were added to an equal volume of RSVLong previously titered to give approximately 115 PFU/25 μl. Serum/virusmixtures were incubated for 2 hours at 37° C. and 5% CO2, to allow virusneutralization to occur, and then 25 μl of this mixture (containingapproximately 115 PFU) was inoculated on duplicate wells of HEp-2 cellsin 96 well plates. After 2 hr at 37° C. and 5% CO2, the cells wereoverlayed with 0.75% Methyl Cellulose/EMEM 5% HI-FBS and incubated for42 hours. The number of infectious virus particles was determined bydetection of syncytia formation by immunostaining followed by automatedcounting. The neutralization titer is defined as the reciprocal of theserum dilution producing at least a 60% reduction in number of synctiaper well, relative to controls (no serum).

2. The Effect of DOTAP Concentration on Immunogenicity

Table 9 shows the effect of DOTAP concentration on the immunogenicity ofthe RSV F antigen in an in vivo cotton rat model. The first twovaccination used the RNA/CNE formulations as shown in Table 9. For thethird vaccination, 3 μg of RSV F subunit protein (in alum) were used forall animals except the naïve group.

TABLE 9 3wp1 3wp2 3wp3 F- F- F- specific specific specific 3wp1 F- 3wp2F- 3wp3 F- total total total specific specific specific RNA IgG IgG IgGNeutralizing Neutralizing Neutralizing Formulation (μg/dose) titerstiters titers IgG titers IgG titers IgG titers 6 μg — 16,373 64,92884,133 327 3,565 3979 F-trimer + Alum 1E6 IU/ — 2819 2,478 15,473 135299 1791 200 ul VRP CNE17 0.01 112 771 23,939 28 66 689 (Ambion 0.1 3511,505 19,495 41 173 1060 MegaScript 1 722 2,379 22,075 82 249 2550 RNA)CMF31 0.01 184 1,015 31,082 31 67 1301 (Ambion 0.1 375 1,250 16,597 5199 2393 MegaScript 1 1013 2,736 20,861 199 341 2783 RNA) 10 4556 6,86727,299 253 672 3593 CMF34 0.01 214 690 25,470 35 38 1440 (Ambion 0.1 4111,574 19,030 45 129 1835 MegaScript 1 953 2,248 18,894 75 353 3224 RNA)10 4,804 5,122 16,566 282 521 3738 CNE17 1 1,042 2,944 23,097 128 2882086 (In house synthesized RNA) Naïve 5 5 5 5 0 10 10 Ambion MegaScriptRNA and in house synthesized RNA were prepared using differentprocesses.

Data from Table 9 show that all CNE-RNA formulations induceddose-dependent immune responses in the hosts (total IgG titers as wellas neutralizing antibody titers). Administering CMF31-RNA and CMF34-RNAformulations produced similar F-specific total IgG titers, and each wasgreater than that of CNE17 at each of the indicated RNA dose. Inaddition, all CNE-RNA formulations induced good neutralizing antibodytiters at 10 μg RNA. Neutralizing antibody titers for the CMF31-RNA,CMF34-RNA, and CNE17-RNA groups were similar, except for surprisinglyhigh titer for the 1 μg RNA/CMF31 group.

Example 5: Assessing the Effects of Buffer Compositions onImmunogenicity

In this example, various emulsions based on CMF34 but with differentbuffer components were prepared.

Table 10 summarizes the results of murine immunogenicity studies whenCMF34-formulated RNAs were prepared using different buffer systems.

TABLE 10 Description 2wp2/ Group N/P 2wp1 # RNA Emulsion ratio 2wp1 2wp2ratio 1 1 μg PBS — 100 2269 23 RSV- F* 2 RV01 PBS — 8388 105949 13 (15)3 1 μg CNE17 with 280 mM 10:1 898 9384 10 RSV- Sucrose 4 F* CMF34 with280 mM 10:1 1835 10853 6 Sucrose 5 CMF34 with 280 mM 10:1 1751 15589 9Sucrose and 1 mM citrate 6 CMF34 with 280 mM 10:1 1699 17078 10 Sucroseand 10 mM citrate 7 CMF34 with 280 mM 10:1 1342 16400 12 Sucrose, 1mMcitrate, and 2 mM NaCl 8 CMF34 with 280 mM 10:1 1318 10467 8 Sucrose, 10mM citrate, and 2 mM NaCl 9 CMF34 with 280 mM 10:1 1735 12457 7 Sucrose,1 mM citrate, and 10 mM NaCl 10 CMF34 with 280 mM 10:1 1365 14414 11Sucrose, 10 mM citrate, and 10 mM NaCl *vA375 replicon.

Example 6: Stability of the Emulsions

Stability of CMF34 was assessed by measuring the average diameter of theemulsion particles and polydispersity after the emulsion was produced(T=0) and after 1 month at 4° C. (T=1 month) and after 2 months at 4° C.(T=1 month). Stability was also assessed after 3, 6 and 12 months at 4°C. The results presented in Table 11 show that the emulsion was stabilefor at least 12 months.

TABLE 11 T = 1 T = 2 T = 3 T = 6 T = 12 T = 0 month months months monthsmonths NanoZS (nm) 101.4 100.6 99.76 99.23 101.0 101.0 Polydispersity0.109 0.102 0.096 0.103 0.080 0.094

Example 7 Immunogenicity of Replicons Encoding Herpes Virus Proteins

A. CMV Proteins

Bicistronic and pentacistronic alphavirus replicons that expressglycoprotein complexes from human cytomegalovirus (HCMV) were prepared,and are shown schematically in FIGS. 1 and 3. The alphavirus repliconswere based on venezuelan equine encephalitis virus (VEE). The repliconswere packaged into viral replicon particles (VRPs), encapsulated inlipid nanoparticles (LNP), or formulated with CMF34. Expression of theencoded HCMV proteins and protein complexes from each of the repliconswas confirmed by immunoblot, co-immunoprecipitation, and flow cytometry.Flow cytometry was used to verify expression of the pentamericgH/gL/UL128/UL130/UL131 complex from pentameric replicons encoding theprotein components of the complex, using human monoclonal antibodiesspecific to conformational epitopes present on the pentameric complex(Macagno et al (2010), J. Virol. 84(2):1005-13). FIG. 2 shows that theseantibodies bind to BHKV cells transfected with replicon RNA expressingthe HCMV gH/gL/UL128/UL130/UL131 pentameric complex (A527). Similarresults were obtained when cells were infected with VRPs made from thesame replicon construct. This shows that replicons designed to expressthe pentameric complex do indeed express the desired antigen and not thepotential byproduct gH/gL.

The VRPs, RNA encaspulated in LNPs, and RNA formulated with CMF34 wereused to immunize Balb/c mice by intramuscular injections in the rearquadriceps. The mice were immunized three times, three weeks apart, andserum samples were collected prior to each immunization as well as threeweeks after the third and final immunization. The sera were evaluated inmicroneutralization assays and to measure the potency of theneutralizing antibody response that was elicited by the vaccinations.The titers are expressed as 50% neutralizing titer.

The immunogenicity of a number of different configurations of abicistronic expression cassette for a soluble HCMV gH/gL complex in VRPswas assessed. FIG. 3 shows that VRPs expressing the membrane-anchored,full-length gH/gL complex elicited potent neutralizing antibodies atslightly higher titers than the soluble complex (gHsol/gL) expressedfrom a similar bicistronic expression cassette. Changing the order ofthe genes encoding gHsol and gL or replacing one of the subgenomicpromoters with an IRES or an FMDV 2A site did not substantially improveimmunogenicity.

To see if bicistronic and pentacistronic replicons expressing the gH/gLand pentameric complexes would elicit neutralizing antibodies indifferent formulations, cotton rats were immunized with bicistronic orpentacistronic replicons mixed with CMF34. Table 12 shows that repliconsin CMF34 elicited comparable neutralizing antibody titers to the samereplicons encapsulated in LNPs.

TABLE 12 Neutralizing antibody titers. The sera were collected threeweeks after the second immunization. 50% Neutralizing Replicon TiterA160 gH FL/gL VRP 10⁶ IU 594 A160 gH FL/gL 1 μg LNP 141 A527 PentamericIRES 1 μg LNP 4,416 A160 gH FL/gL 1 μg CMF34 413 A527 Pentameric IRES 1μg 4,411 CMF34

B. VZV Proteins

Nucleic acids encoding VZV proteins were cloned into a VEE repliconvector to produce monocystronic replicons that encode gB, gH, gL, gE,and gI, and to produce bicistronic replicons that encode gH/gL or gE/gI.In the bicistronic replicons, expression of each VZV open reading framewas driven by a separate subgenomic promoter.

To prepare replicon RNA, plasmid encoding the replicon was linearized bydigestion with PmeI, and the linearized plasmid was extracted withphenol/chloroform/isoamylalchohol, precipitated in sodiumacetate/ethanol and resuspended in 20 μl of RNase-free water.

RNA was prepared by In vitro transcription of 1 μg of linearized DNAusing the MEGAscript T7 kit (AMBION# AM1333). A 20 μl reaction was setup according to the manufacturer's instruction without cap analog andincubated for 2 hours at 32° C. TURBO DNase Out) was added and themixture was incubate for 30 min. at 32° C. RNase-free water (30 μl) andammonium acetate solution (30 μl) were added. The solution was mixed andchilled for at least 30 min at −20° C. Then the solution was centrifugedat maximum speed for 25 min. at 4° C. The supernatant was discarded, andthe pellet was rinsed with 70% ethanol, and again centrifuged at maximumspeed for 10 min. at 4° C. The pellet was air dried and resuspended in50 μl of RNase-free water. The concentration of RNA was measured andquality was check on a denaturing gel.

The RNA was capped using the ScriptCap m7G Capping System (Epicentre#SCCE0625). The reaction was scaled by combining the RNA and RNase-freewater. The RNA was then denatured for 5-10 min. at 65° C. The denaturedRNA was transfered quickly to ice and the following reagents were addedin the following order: ScriptCap Capping Buffer, 10 mM GTP, 2 mM SAMfresh prepared, ScriptGuard RNase inhibitor, and ScriptCap CappingEnzyme. The mixture was incubated for 60 min. at 37° C. The reaction wasstopped by adding RNase-free water and 7.5 M LiCl, mixing well andstoring the mixture for at least 30 min at −20° C. Then, the mixture wascentrifuged at maximum speed for 25 min. at 4° C., the pellet was rinsedwith 70% ethanol, again centrifuged at maximum speed for 10 min. at 4°C. and the pellet was air dried. The pellet was resuspended inRNase-free water. The concentration of RNA was measured and quality waschecked on a denaturing gel.

RNA Transfection

Cells (BHK-V cells) were seeded on 6-well plates brought to 90-95%confluence at the time of transfection. For each transfection 3 μg ofRNA was diluted in 50 mL OPTIMEM media in a first tube. Lipofectamine2000 was added to a second tube contained 50 mL OPTIMEM media. The firstand second tubes were combined and kept for 20 min. at room temperature.The culture media in the 6-well plates were replaced with fresh media,and the RNA-Lipofectamine complex was placed onto the cells, and mixedby gently rocking the plate. The plates were incubated for 24 hours at37° C. in a CO₂ incubator.

For immunofluorescence, transfected cells were harvested and seeded in96 well plate, and intracellular staining was performed usingcommercially available mouse mAbs (dilution range 1:100 1:400). Cellpellets were fixed and permeabilized with Citofix-Citoperm solutions. Asecondary reagent, Alexa488 labelled goat anti-mouse F(ab′)₂ (1:400final dilution), was used.

Expression of VZV proteins gE and gI was detected in cells transfectedwith monocistronic constructs (gE or gI), and expression of both gE andgI was detected in cells transfected with a bicistronic gE/gI constructin western blots using commercially available mouse antibodies, 13B1 forgE and 8C4 for gI. Expression of VZV protein gB was detected in cellstransfected with a monocistronic construct encoding gB, byimmunofluorescence using commercially available antibody 10G6.Expression of the VZV protein complex gH/gL, was detected byimmunofluorescence in cells transfected with monocistronic gH andmonocistronic gL, or with a bicistronic gH/gL construct. The gH/gLcomplex was detected using commercially available antibody SG3.

Murine Immunogenicity Studies

Groups of 8 female BALB/c mice aged 6-8 weeks and weighing about 20 gwere immunized intramuscularly with 7.0 or 1.0 μg of replicon RNAformulated with CMF32 or LNP (RV01) at day 0, 21 and 42. Blood sampleswere taken from the immunized animals 3 weeks after the 2nd immunizationand 3 weeks after the 3rd immunization. The groups are shown in Table13.

TABLE 13 Group Antigen Dose (micrograms) Formulation 1 YFP 7 CMF32 2 YFP1 CMF32 3 gB 7 CMF32 4 gB 1 CMF32 5 gE 7 CMF32 6 gE 1 CMF32 7 gH 7 CMF328 gH 1 CMF32 9 gI 7 CMF32 10 gI 1 CMF32 11 gL 7 CMF32 12 gL 1 CMF32 13gE/gI 7 CMF32 14 gE/gI 1 CMF32 15 gH/gL 7 CMF32 16 gH/gL 1 CMF32 Immuneresponse to VZV antigens

Serum samples were tested for the presence of antibodies to gB, byintracellular staining of VZV-replicon transfected MRC-5 cells. MRC-5cells were maintained in Dulbecco Modified Eagle's Medium with 10% fetalbovine serum. VZV Oka strain inoculum (obtained from ATCC) was used toinfect MRC-5 cell culture and infected whole cells were used forsubpassage of virus. The ratio between infected and un-infected cellswas 1:10. 30 hrs post infection cells were trypsin-dispersed for seedingin a 96 well plate to perform an intracellular staining with pools ofmice sera (dilution range 1:200 to 1:800) obtained after immunization.Commercial mAbs were used as controls to quantify the infection level.Cell pellets ware fixed and permeabilized with Citofix-Citopermsolutions. A secondary reagent, Alexa488 labelled goat anti-mouseF(ab′)₂ was used (1:400 final dilution).

Commercial antibodies to gB (10G6), gH (SG3), and gE (13B1 (SBA) and8612 (Millipore)) were used as positive controls, and eachintracellularly stained infected MRC-5 cells Immune sera obtained 3weeks after the third immunization with either 1 or 7 μg of RNAformulated with CMF32 were diluted 1/200, 1/400 and 1/800 and used tointracellularly stain infected MRC-5 cells. The results are shown inFIG. 4 (Study 1, groups 1, 5, 7, 9, 11, 13 and 15, CMF32 formulation).

Neutralizing Assay

Each immunized mouse serum was serially diluted by two fold incrementsstarting at 1:20 in standard culture medium, and added to the equalvolume of VZV suspension in the presence of guinea pig complement. Afterincubation for 1 hour at 37° C., the human epithelial cell line A549,was added. Infected cells can be measured after one week of culture bycounting plaques formed in the culture under microscope. From the plaquenumber the % inhibition at each serum dilution was calculated. A chartfor each serum sample was made by plotting the value of % inhibitionagainst the logarithmic scale the dilution factor. Subsequently anapproximate line of relationship between dilution factor and %inhibition was drawn. Then the 50% neutralization titer was determinedas the dilution factor where the line crossed at the value of 50%inhibition.

Table 14 shows that sera obtained from mice immunized with monocistronicgE, bicistrnic gE/gI, and bicistronic gH/gL contained robustneutralizing antibody titers.

TABLE 14 Neutralization titers of pooled sera from mice immunized with 7μg RNA in CMF32 Con- Mouse trol ID (YFP) gB gE gI gE/gI gH gL gH/gL 1<20  <20    1111 <20     440 <20  <20    1070 2 <20  <20     413  51  >2560 <20  <20  >2560 3 <20  <20  >2560 <20    1031 <20 <20  >2560 4 <20    20    2128 <20    1538 <20  <20  >2560 5 <20    20    861 <20     636   20  <20  >2560 6 <20  <20    1390 <20    2339 <20 <20  >2560 7 <20  <20     969 <20    1903 <20  <20     900 8 <20  <20   1011   20    1969   20  <20  >2560 9 <20* <20*   <20* <20*   <20* <20*<20*   <20* *pre-immune pooled sera

Example 8: The Solubility of Fatty Acids in Squalene

In this Example, the solubility of various fatty acids in squalene wasexamined, and shown in Table 15. Fatty acids at indicated amounts (40,20, 10, or 5 mg/mL,) were mixed with squalene at 60° C. In Table 15, (√)means that the fatty acid was soluble in squalene at the specifiedconcentration; “x” means that the fatty acid was not soluble in squaleneat the specified concentration; and “-” means that the solubility of thefatty acid at the specified concentration was not tested (because thefatty acid was soluble at a higher concentration). After the fatty acidswere dissolved in squalene, the solutions were left at 4° C. overnight.The column labeled 4° C. overnight shows the solubility of the solutionsin which each fatty acid was at its top concentration. For example oleicacid was soluble in squalene at 40 mg/ml and remained soluble insqualene at 4° C. overnight.

TABLE 15 40 20 10 5 4° C. mg/ mg/ mg/ mg/ (over- Fatty acid mL mL mL mLnight) Saturated Undecanoic Acid √ — — — √ Fatty Acids Tridecanoic Acid√ — — — x (Odd Pentadecanoic Acid √ — — — x Carbon Heptadecanoic Acid xx x √ x Chains) Nonadecanoic Acid x x x x x Heneicosanoic Acid x x x x xTricosanoic Acid x x x x x Saturated Capric acid (10:0) √ — — — √ FattyAcids Lauric acid (12:0) √ — — — x (Even Myristic Acid (14:0) x √ — — xCarbon Palmitic Acid (16:0) x x x √ x Chains) Stearic Acid (18:0) x x x√ x Arachidic Acid (20:0) x x x √ x Behenic Acid (22:0) x x x x xLignoceric Acid (24:0) x x x x x Unsaturated Docosahexaenoic Acid √ — —— √ fatty acids (22:6) Elaidic Acid (18:1)- √ — — — √ trans Erucic Acid(22:1) √ — — — √ Linoleic Acid (18:2) √ — — — √ Linolenic Acid (18:3) √— — — √ Nervonic Acid (24:1) √ — — — x Oleic Acid (18:1)-cis √ — — — √Palmitoleic Acid (16:1) √ — — — √ Petroselinic Acid (18:1) √ — — — √Sequences

The nucleotide sequence of a DNA encoding the vA317 RNA, which encodesthe RSV-F antigen (SEQ ID NO: 1).

The nucleotide sequence of a DNA encoding the vA142 RNA (SEQ ID NO: 2).

The nucleotide sequence of a DNA encoding the vA375 RNA (SEQ ID NO: 3).

A526 Vector: SGP-gH-SGP-gL-SGP-UL128-2A-UL130-2Amod-UL131 (SEQ ID NO:4).

A527 Vector: SGP-gH-SGP-gL-SGP-UL128-EMCV-UL130-EV71-UL131 (SEQ ID NO:5).

A531 Vector: SGP-gHsol-SGP-gL (SEQ ID NO: 6).

A532 Vector: SGP-gHsol-2A-gL (SEQ ID NO: 7).

A533 Vector: SGP-gHsol-EV71-gL (SEQ ID NO: 8).

A534 Vector: SGP-gL-EV71-gH (SEQ ID NO: 9).

A535 Vector: SGP-342-EV71-gHsol-2A-gL (SEQ ID NO: 10).

A536 Vector: SGP-342-EV71-gHsol-EMCV-gL (SEQ ID NO: 11).

A537 Vector: SGP-342-EV71-gL-EMCV-gHsol (SEQ ID NO: 12).

A554 Vector: SGP-gH-SGP-gL-SGP-UL128-SGP-UL130-SGP-UL131 (SEQ ID NO:13).

A555 Vector: SGP-gHsol-SGP-gL-SGP-UL128-SGP-UL130-SGP-UL131 (SEQ ID NO:14).

A556 Vector: SGP-gHsol6His-SGP-gL-SGP-UL128-SGP-UL130-SGP-UL131 (SEQ IDNO: 15).

VZV gB (SEQ ID NO: 16).

VZV gH (SEQ ID NO: 17).

VZV gL (SEQ ID NO: 18).

VZV gI (SEQ ID NO: 19).

VZV gE (SEQ ID NO: 20).

VZV VEERep.SGPgB (SEQ ID NO: 21).

VZV VEERep.SGPgH (SEQ ID NO: 22).

VZV VEERep.SGPgL (SEQ ID NO: 23).

VZV VEERep.SGPgH-SGPgL (SEQ ID NO: 24).

VZV VEERep.SGPgE (SEQ ID NO: 25).

VZV VEERep.SGPgI (SEQ ID NO: 26).

VZV VEErep.SGPgE-SGPgI (SEQ ID NO: 27).

The specification is most thoroughly understood in light of theteachings of the references cited within the specification. Theembodiments within the specification provide an illustration ofembodiments of the invention and should not be construed to limit thescope of the invention. The skilled artisan readily recognizes that manyother embodiments are encompassed by the invention. All publications andpatents cited in this disclosure are incorporated by reference in theirentirety. To the extent the material incorporated by referencecontradicts or is inconsistent with this specification, thespecification will supersede any such material. The citation of anyreferences herein is not an admission that such references are prior artto the present invention.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following embodiments.

The invention claimed is:
 1. A composition comprising an RNA moleculeand a stable oil-in-water emulsion comprising particles that aredispersed in an aqueous continuous phase, wherein the average diameterof said particles is from about 80 nm to about 150 nm; wherein theaverage diameter does not change by more than 10% when the emulsion isstored at 4° C. for one month; wherein the emulsion comprises an oil anda cationic lipid, wherein the head group of the cationic lipid comprisesa quaternary amine; and wherein: (i) the ratio of oil:lipid (mole:mole)in the oil-in-water emulsion is at least about 8:1 (mole:mole); (ii) theconcentration of cationic lipid in said composition is at least about1.25 mM; (iii) the cationic lipid is not DC-Cholesterol, and (iv) theRNA molecule is complexed with a particle of the oil-in-water emulsion.2. The composition of claim 1, wherein the average diameter of saidparticles is from about 80 nm to about 130 nm.
 3. The composition ofclaim 1, wherein said composition has an N/P ratio of at least about4:1.
 4. The composition of claim 1, wherein the ratio of oil:lipid(mole:mole) is from about 10:1 (mole:mole) to about 43:1 (mole:mole). 5.The composition of claim 1, wherein said oil-in-water emulsion comprisesfrom about 0.1% to about 5% (w/v) oil.
 6. The composition of claim 5,wherein said oil is squalene or squalane.
 7. The composition of claim 1,wherein said particles further comprise a surfactant.
 8. The compositionof claim 7, wherein said surfactant is a nonionic surfactant.
 9. Thecomposition of claim 7, wherein said surfactant is not a PolyethyleneGlycol (PEG)-lipid.
 10. The composition of claim 7, wherein the cationicoil-in-water emulsion comprises from about 0.005% to about 1.25% (v/v)surfactant.
 11. The composition of claim 7, wherein said surfactant isSPAN85 (Sorbitan Trioleate), Tween 80 (polysorbate 80), or a combinationthereof.
 12. The composition of claim 11, wherein the cationicoil-in-water emulsion comprises about 0.25% (v/v) Tween 80 and about0.25% (v/v) SPAN85.
 13. The composition of claim 1, wherein saidcationic lipid is selected from the group consisting of:1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP),1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC),N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), and N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).
 14. Thecomposition of claim 13, wherein said cationic lipid is DOTAP.
 15. Thecomposition of claim 14, wherein the concentration of DOTAP in saidcomposition is at least about 1.29 mM (0.9 mg/mL).
 16. The compositionof claim 1, wherein the RNA molecule is a self-replicating RNA moleculethat encodes an antigen.
 17. The composition of claim 16, wherein theself-replicating RNA is an alphavirus-derived RNA replicon.
 18. Thecomposition of claim 1, wherein the RNA molecule is a polycistronic RNAencoding two or more antigens.
 19. The composition of claim 18, whereinthe polycistronic RNA is a alphavirus replicon.
 20. The composition ofclaim 19, wherein the alphavirus replicon contains a first nucleotidesequence encoding a first antigen and a second nucleotide sequenceencoding a second antigen, wherein the first nucleotide sequence and thesecond nucleotide sequence are operably linked to control elements. 21.The composition of claim 20, wherein the first nucleotide sequence isoperably linked to a first control element and the second nucleotidesequence is operably linked to a second control element.
 22. Thecomposition of claim 20, wherein the control elements are independentlyselected from the group consisting of a subgenomic promoter, an IRES,and a viral 2A site.
 23. A method of generating an immune response in asubject, comprising administering to a subject in need thereof thecomposition of claim
 1. 24. The composition of claim 1 furthercomprising an antioxidant.